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How would one track a mineral nutrient in a plant in order to prove that nutrient has been re mobilized?

How would one track a mineral nutrient in a plant in order to prove that nutrient has been re mobilized?



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Before abscission - senescence of a plant structural components that contain mineral nutrients (E.g. Magnesium, Potassium… ) are re-mobilized from the senescent tissue and used in other plant tissue for anabolic activities.

Could we design an experiment to prove this?

I was told that radioisotope method would work. We can detect the presence of the radioactive particle in one part at a time-point, but how would we know whether it is the very same particle that shows up elsewhere after abscission?


It is not possible to track individual molecules within a plant. However, one can track the level of radioactivity from place to place. So, in the case you are talking about, one would find that some level of radioactivity persisted for a time at one location, then it is observed to decline while it increases in another. There also might be a delectably increased level of radioactivity along the pathway between those two areas - regardless, one would logically connect decreased activity in one area with increased activity in another.


Magnesium isotopes would allow you to track the movement of magnesium.

Injection of the radioisotope 28Mg (half-life of 20.915 hours) into senescent cells may allow you to observe the direct effect of magnesium movement from senescent cells to other cells. If abscission were to occur, you could check abscised plant tissue. If you wanted to check if anabolism was also occurring, this may take more thought.

This is assuming senescents and abscission in the plant model takes under ~20 hours. Which I think is unlikely, however there are plenty of other 'mineral nutrient' and magnesium radioisotopes available.

Note
Radioactive isotopes have the ability to emit radiation allowing them to be imaged. This is great when they are inorganic molecules, because plant tissue is (mostly) organic. These variables allow imaging to be highly effective at particular emissions.

A similar approach was done here (Pay-wall(another here with-out paywall)). However these studies added another step 'fractionation'. This may be a helpful idea, fractionation is a separation process to measure the abundance of Magnesium (they refer to it as Magnesium Purification). This is another reason to use an isotope, instead of imaging it may be useful for purification.


Energy and Nutrient Recovery from Swine Manures

While the primary method of swine manure management in the United States is temporary storage followed by land application as crop fertilizer, there is increasing interest in recovering energy and nutrients from manures prior to land application. Insufficient nutrient assimilation capacity in nearby crop land, or interest in adding value to swine manure beyond the fertilizer value, are among the reasons that alternative management strategies may be sought. Producers who consider alternative manure uses will find many options available. This publication describes several energy and nutrient recovery processes currently available. Each process is explained and primary issues that a producer should consider with each process are discussed.

Objectives

  • Summarize processes that extract energy from animal manures (urine and/or feces) as a mechanism to recover value from manure by providing on- or off-site energy.
  • Summarize the economic feasibility of energy recovery and describe the non-economic benefits that may accompany several of these technologies.
  • Summarize opportunities and approaches that enhance the ability to recover nutrients in order to avoid over-application of nutrients to cropland.

Energy Recovery: Introduction

Processes that extract energy from animal manures (urine and/or feces) have been considered as a mechanism to recover value from manure by providing on- or off-site energy. The feasibility of energy recovery from manures is tied to the energy and labor costs at the location in question, as well as to the non-economic benefits that may accompany several of these technologies (i.e., odor control). While gross energy in most animal feedstuffs varies very little, there may be large variation in the amount of energy that can be digested and utilized by animals, and hence available in the manure. For example, cellulose is undigested by non-ruminants that and undigested carbon sources in any ration pass out of the digestive tract as a potential energy resource. Lignin in feeds will burn but is unavailable to animals, even ruminants, and is relatively unavailable to anaerobic microbes. The manure management system used also influences the energy value of manure. For example, the gross energy value of manures that have been highly diluted with water would be less than undiluted manures. This does not mean that energy recovery is not feasible with liquid manure slurries. Some energy recovery options, such as anaerobic digestion, are best suited to liquid manures.

Why Consider Anaerobic Digestion?

The anaerobic digestion of swine manure produces biogas (primarily a combination of methane and carbon dioxide) that can be burned to recover energy. Biogas produced from the anaerobic digestion of swine manure typically has a heating value of around 600 Btu / ft3 and is composed of about 65% methane [1]. The methane contained in the biogas generated by the anaerobic digestion process can be burned as fuel to either generate electricity or to provide heat for on farm-use, or for both, which is referred to as cogeneration. In addition to the potential for energy recovery, when properly operated, anaerobic digestion of manure can also provide significant odor reduction benefits when compared to traditional manure management methods.

How Anaerobic Digestion Works

Anaerobic digestion is the breakdown of organic compounds contained in manure by microorganisms without the presence of oxygen. Anaerobic degradation begins in the lower digestive tract of animals and continues in feces droppings, manure piles, and storage facilities. Anaerobic digestion involves multiple steps and several classes of bacteria. While methane and carbon dioxide are odorless, intermediate compounds formed during anaerobic digestion have odors. Because of this, if the anaerobic digestion process is disrupted and not completed odorous intermediate compounds may be emitted. Excess biogas produced is typically burned using a flare because the storage of the gas would require compression and is not typically economically feasible.

Anaerobic digesters can be divided into two basic types, suspended growth and fixed film. As the name implies, the consortium of anaerobic bacteria degrading the waste are suspended in the manure slurry in suspended growth systems. The suspended growth digester configurations most often used with animal waste include covered lagoons, mixed digesters (commonly called CSTR for continuously stirred tank reactors), plug-flow, and ASBRs (anaerobic sequencing batch reactors). The majority of anaerobic digesters used with swine manure are covered lagoons and CSTRs. Plug-flow digesters require a high solids manure (12-14% total solids) and are used extensively with dairy waste, but are not appropriate for most swine manures. While ASBRs are appropriate for swine manures, the use of this system type at the farm-scale has been very limited.

The bacteria in fixed film systems are attached to some type of support media, such as plastic pipes or other structure, where the bacteria can grow. Fixed film systems are commonly called anaerobic filters, since the waste must “filter” through the support media in the digester. The support media in fixed film animal waste digesters is designed to have large openings (usually 3 inches or greater) in order to prevent plugging these openings with manure solids. Fixed film digesters are best suited for low solids manures, like those commonly associated with flush manure management systems. Because bacteria remain attached, fixed film systems are capable of digesting greater volumes of manure per unit time than other types of digester systems, thereby reducing the size of the digester. Digestion systems can be designed to operate at ambient temperature or heated to operate at higher temperatures to increase the waste digestion rate. Biogas may be converted to energy to either heat the digester or provide some portion of the operation’s energy needs.

Hydraulic retention time (HRT) refers to the time that influent material spends within a reactor, and can be computed by the following expression: HRT = V/Q, where V is the reactor volume (e.g., cubic feet), and Q is the flow rate into the reactor (e.g., cubic feet per day). Because HRT is proportional to reactor volume it has the largest influence on the fixed costs of a digester. Shorter HRT and higher loading rates typically decrease cost, but they also decrease the extent of VS conversion to biogas. Digester systems are often designed based on volatile solids (VS) loading rates. For example, a suggested range of loading rates for anaerobic digestion of swine wastes is 0.24 to 0.50lb VS/ft3/d.

As a rule-of-thumb, 5.6ft3 of methane can be generated for each pound of volatile solids (VS) destroyed [2]. For the typical finishing pig:

84 lb VS/finished animal X 50% VS conversion efficiency X 5.6ft3 /lb VS =

1,760ft3 of methane per finished pig

To get a better feel for the amount of energy that could be generated from swine manure we can estimate the amount of electricity that could be produced from the manure from a 150 lb finishing hog during a one-day period. If we burn the biogas in an internal combustion engine to generate electricity the manure from a 150lb finish hog would provide about 10 watts of continuous power. If we were producing power from swine manure by burning the biogas from an anaerobic digester in an internal combustion generator we would need the manure from ten 150 pound finish hogs to keep a 100 watt light bulb burning.

Considerations

Over the past three decades around 100 full-scale animal manure digester systems have been installed on farms in the United States. It is estimated that at least 50% of these digesters are no longer operational. However, the failure rate has deceased over time suggesting that current systems may be more reliable than earlier ones. Daily monitoring and management is essential to keep the system running. The consistent allocation of labor to manage the digester is necessary for success.

Anaerobic digestion stabilizes manure against further decomposition, reduces odorous intermediates, reduces the manure carbon content through CO2 and methane production, and maintains most of the nutrient value of the manure (much now in bacterial cells) for land application. Ammoniacal nitrogen is increased and, if not lost to the atmosphere, is readily available for nitrification and crop uptake. Because manure nutrient value is retained, producers who are land-limited must seek additional means of removing manure nutrients.

The potential to capture and utilize biogas as an alternate energy source is a benefit of these systems. Because the manure generation rate varies over the swine life cycle, many times the power generation rate and power use rate on the farm will not match. One option is to sell excess power to the local utility. In some locations a premium rate for “green” power may be paid for this electricity and in other locations a rate far below the rate charged by the utility may be paid. In either case it should be noted that the interconnection with a utility power system can be very complicated due to issues concerning synchronization and safety. Because of these issues, many farms opt to flare biogas that is produced in excess of the farms power needs.

Thermochemcial Conversion: Why Consider Gasification or Liquefaction?

Thermochemcial processes such as gasification or liquefaction can be used to convert suitable manures into gases or liquids that can be used directly as low to medium BTU value fuels, or further processed into higher BTU value fuels.

How Gasification Works

Wastes containing organic matter can be thermochemically reformed into alternative energy sources. The thermochemcial conversion processes of gasification take place in a heated enclosure, absent of oxygen [3]. Gasification is a process where organic materials such as manures undergo oxygen deficient thermal decomposition into gases with low to medium BTU value. The primary gases produced include methane, carbon monoxide and hydrogen.

The main obstacle for converting swine manure into a suitable feedstock for gasification is moisture content [4,5]. Commonly employed waste management systems at swine facilities have a waste stream that is too wet for this system to be economically feasible. Some researches are studying scenarios where the waste management system is altered such that the waste material to undergo gasification is drier than material from traditional slurry systems [4].

How Liquefaction Works

Liquefaction is a high-pressure hydrogenation process where organic materials are liquefied in an oxygendeficient environment. The liquefaction process converts the organic material into a tar or oil using a sequence of physical and chemical processes. Liquefaction increases the hydrogen to carbon ratio of the product (the liquid fuel) relative to that present in the manure. Unlike gasification, this process requires sufficient moisture for completion, as well as addition of a reducing gas such as H2 or CO [3,6]. While some research on liquefaction of swine manure has taken place, currently, there are no commercial or pilot scale liquefaction facilities using animal manure as feedstock in place [6].

Considerations

While a great deal of research is currently being conducted concerning the gasification and liquefaction of animal manures into gases and liquid fuels, no full-scale use of this technology with animal waste is currently in use. Several pilot-scale operations, however are in operation. In order to be successfully gasified, swine waste slurries would require drying, or the manures would need to be collected in a relatively dry state.

Why Consider Direct Combustion?

Dry manures can be burned as a direct fuel source, or combined with other materials such as wood or coal. Fuels derived either wholly or partially from manures can be burned directly to produce heat and generate electricity. However, because swine produce relatively wet manure, this technology has not been pursued for swine wastes.

How Direct Combustion Works

Manure, in a relatively dry form, may be burnt directly as fuel. The use of manure as fuel is an ancient practice that is still utilized in many developing countries. The gross energy of manure, for burning, varies little between species and most of that variability can be explained by differences in ash content. Manure energy content is approximately 27.0Mcal in 15lb dry matter (DM). At 90% DM (air-dry), this represents about half that of coal.

Considerations

Most swine manure is too wet to consider burning. However, for systems that produce a dry manure, advantages of combustion processes include energy generation that adds value to manure, retention of nutrients (P and K) for fertilizer, reduction of storage requirements for the retained nutrients, and loss of N during the combustion process in the event that N loss is desirable.

When manure is burnt, the ash nutrients still need to be managed accountably. However, the responsibility for managing the ash and its nutrients (P and K, included) may be transferred depending on the individual arrangements between the burning facility and the livestock operation. Economics has been the single largest contributing factor to the limited burning that has occurred. However, other considerations include biosecurity implications of hauling to a centralized facility. Also, the exhaust gases need to be purified by scrubbers, cyclones and other types of high performance filters.

Energy Recovery Summary

Swine manure contains energy that can be recovered by various processes. The anaerobic digestion of manure to generate biogas is the process that is most easily adapted to liquid swine waste slurries. While the generation of electricity from biogas derived from animal manures is completely feasible, the economic feasibility of this system depends on subsidies paid for green power in many regions of the United States. The primary reason many animal producers give for operating anaerobic digesters is odor control. In many cases all of the biogas generated by these systems is simply flared.

Other energy recovery options are feasible for dry manures. Direct combustion and gasification both use solid manures as a fuel source. In the gasification process dry manures are thermochemically converted into low to medium BTU value gases that can be directly burned as fuel, or further processed into higher BTU value fuels such as ethanol. Economics of these systems have contributed to lack of widespread adoption.

Given the options available, when making a decision regarding the potential to recover energy from manure producers must weigh 1) their need for nutrient conservation, 2) their own need for energy that may be generated and their opportunities to sell excess energy, with 3) the overall economics of the various options relative to their goals and objectives.


Blood is analyzed in a lab. The blood is put into a centrifuge and spun until it separates.

Reference ranges

Then, technicians compare results to a “reference range.” The reference range is the range of expected values for each test listed.

Expected ranges used in lab analyses include 95% of the “healthy” population. So, 95% of healthy people would have lab values within these ranges. This range varies depending on the lab, region (e.g. US vs EU) and type of blood component.

A number above or below this reference range can give valuable diagnostic information about body systems. High and low values are especially useful when taken in context with other symptoms, lifestyle factors, and tests.

Variation in results and ranges

Lab values vary for each person and must be assessed relative to other factors. While reference ranges are established after testing a large number of healthy people, everyone is slightly different.

Blood analyses vary based on:

  • Time of year
  • Posture/positioning
  • Food/fluid intake
  • Stress
  • Medication/supplement use
  • Alcohol
  • Smoking
  • Exercise/physical activity
  • General tests

How to grow your own science experiment

Does fertilizer make plants bigger? My editor and I grew radishes to find out. Spoiler: Ours did not look this good.

Nastco/iStock/Getty Images Plus

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December 9, 2020 at 6:30 am

This article is one of a series of Experiments meant to teach students about how science is done, from generating a hypothesis and designing an experiment to analyzing the results with statistics. You can repeat the steps here and compare your results — or use this as inspiration to design your own experiment.

Sometimes when you tend a garden, your plants end up looking oddly sad. Maybe they’re short and stubby, or not as leafy as you’d like. The first thing some people might suggest is to add a little fertilizer to make your plants bigger and taller. But will fertilizer do that? Here’s an experiment to find out.

Explainer: The fertilizing power of N and P

Plants are marvels. Using carbon dioxide, light and water, they can make sugar out of (almost) thin air. “Most of a plant is made from carbon dioxide,” explains Jessica Savage. “A lot of times people think the plant grows or is built out of things from the soil. But it’s growing out of the air.” As a botanist, Savage studies plants. She works at the University of Minnesota in Duluth.

Plants can’t quite survive on air alone. They do need a few other elements. For example, the backbone of DNA — the molecule with the plant’s genetic instructions — has phosphorus atoms in it. So does ATP, the chemical that helps transfer energy around a cell. Proteins — molecules that do much of a cell’s work — need nitrogen atoms.

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Usually, plants get nitrogen and phosphorus from the soil. Some plants are known as nitrogen-fixing. They can pull nitrogen from the air and transform it into nitrogen-containing molecules that plants can use. But most plants can’t do this. They have to rely on other plants or fungi to transform nitrogen for them. They also have to get phosphorus in the form of phosphate (phosphorus bound to four oxygen atoms), which is broken down from rocks in the earth.

Soils have plenty of nitrogen and phosphorus in them. But many do not. Gardening fertilizer contains nitrogen and phosphorus in forms that plant roots can easily slurp up. With all the extra nutrients, the fertilizer ads say, plants will grow bigger and faster.

Explainer: How photosynthesis works

“If [plants] are given a lot of light and nitrogen, they might increase chlorophyll and photosynthesis,” Savage says. That might mean the plants end up with more leaves. With more leaves, she notes, they’ll have more sugar. Those sugars can be made into more plant materials. With fertilizer, Savage explains, plants should get bigger, because they’ll make more sugar.

The question is whether the fertilized plants will have bigger roots, bigger leaves or both. “Will they focus on growing above or below ground?” she asks.

That’s a hypothesis I can test. My hypothesis is that fertilized plants will be bigger than those that are not fertilized.

You don’t need too much stuff for this experiment. A good notebook, small pots, radish seeds, organic potting soil, fertilizer and a spot with plenty of sun. B. Brookshire

Grow radish, grow

I bought several packs of seeds, 24 small plastic seed pots, plant fertilizer and potting soil. I made sure the soil didn’t contain added fertilizer.

I wanted something that I could grow quickly, that wouldn’t take up a lot of space and that wouldn’t get too big. I ran this experiment in early fall in Maryland. So I knew I needed a plant that could grow when it’s cool. I picked radishes, which grow well in the early fall or spring. Some varieties can grow a full radish in only 21 days.

  1. Radish seeds are VERY tiny. Keep track to make sure you don’t lose any. B. Brookshire
  2. Make a small hole about the depth of your fingertip, push in the seed and cover (left). You also want to keep your plants together, so they get the same amount of sun and rain. Label them all so you know which is fertilized and which is not. B. Brookshire

I kept 12 of the pots and one pack of seeds for myself. I gave the other 12 pots and the other packet of seeds — along with some fertilizer and soil — to my editor, Sarah Zielinski. This was to provide an additional control for location. After all, what if my yard just happens to be much better for growing plants? What if it’s worse? By splitting the plants up between my yard and Sarah’s, I hoped to make sure that any difference with the plants came from the fertilizer.

Sarah and I planted our seeds. Sometimes, seeds don’t sprout. So we carefully planted four evenly spaced seeds in each pot. Six of my pots (and six of Sarah’s) served as controls — pots that would not get fertilizer. Our other six were treated with fertilizer. For each of us, this added up to 24 control seeds, and 24 seeds that would get fertilizer.

It’s important to read and follow the instructions for the type of fertilizer you use. (Mine required mixing a tiny capful of liquid with five gallons of water.) Too much can cause fertilizer burn, where plants brown or even die. That’s because the nitrogen in fertilizer mix is in the form of a salt called ammonium nitrate. Such salts in the soil can cause water to leave the plant and head toward the salty soil — a process called osmosis. This can make the plant dry out and look burned.

We watered all the plants equally with clean water every other day (unless it rained). Once a week, we applied fertilizer to half the pots. We also took pictures every day, so we could see the plants change over time.

As I expected, many of our seeds didn’t sprout. In fact, only about a fourth of mine sprouted. Sarah has a greener thumb. She successfully grew half of hers.

Radishing results

The radishes were still too small to weigh, so we measured the root and leaf length. B. Brookshire

Day 21 was the moment of truth! Sarah and I pulled out the radishes, weighed them and measured the leaves and roots.

I pulled out my first radish — and was pretty disappointed.

While these plants can mature in 21 days, that doesn’t mean they always do. Our radishes were pretty puny. But that’s not a bad thing. After all, if all the radishes had grown until they couldn’t get any bigger, it might be harder to see any differences from the fertilizer.

Unfortunately, the radishes were so small that they weighed less than one gram. Most home kitchen scales don’t measure masses that small. Sarah and I were stuck measuring the length of the roots and leaves to see if there was any difference.

We started by counting the leaves on each plant. Together, we grew a total of 30 plants that received no fertilizer. These control plants had an average of 4.1 leaves. We also grew 24 plants in our fertilized pots. These had an average of 5.3 leaves. It seems the fertilized plants had more leaves than control plants.

The control radish (left) looks a good bit smaller than the fertilized one on the right. But does the difference matter? Always grow more than two radishes, enough to do statistics. B. Brookshire

But that doesn’t mean the difference was due to the fertilizer. To find that out, I need to run statistics — tests I can use to interpret my data. In this case, we have two groups — fertilized and control. I used a t test, which can be used to compare two groups to each other. There are lots of sites online that will let you copy and paste in your data. I used this one from GraphPad.

A t test gives you a p value. A p value is a measure of the probability that just by chance I would see a variation between the groups as big as the one I measured. Usually, it’s expressed as a decimal, such as 0.05. That would be a five percent likelihood that I would get a difference as big or bigger than the one I saw if there was no real difference between the groups. Scientists often consider p values smaller than 0.05 to be meaningful — what they call statistically significant.

In this case, the p value between the fertilized and control leaves was 0.0001, or 0.01 percent. That difference is statistically significant. But that doesn’t tell you if the difference between the two is a big one. A difference can be very small and still be statistically significant. To find out if I have a big difference, I need to run a test called a Cohen’s d. You can also run that for free online. I used the calculator here.

This is a table that shows the results of my statistics. The top two rows are the average number of leaves, average root length and average leaf length for control (row one) and fertilized plants (row two). The p value from the t tests comparing the numbers is row three, and the Cohen’s d is row four. You can see that all of my results were statistically significant. But only the leave count and leaf length differences were large.

For the Cohen’s d calculation, I need a number called the standard deviation. This is the amount by which each set of data differs from the mean (or average). To find that, I went to my data in Microsoft Excel, typed in the function “= STDEV” and highlighted my data set. I plugged into my calculator the mean, the standard deviation and the number of plants in each group.

My Cohen’s d was 1.3. Scientists usually consider any number over 0.8 to be a large difference. So it appears that our fertilized plants had more leaves than our non-fertilized controls, and that the fertilizer made a big difference in the number of leaves.

We also measured the length of the leaves and the length of the roots. I’ve included the p values for each one and the Cohen’s d in the table below. Fertilized plants had longer roots, but that difference was not big. They also had longer leaves, and here the difference was again large.

  1. Fertilized plants (right, yellow) had more leaves after three weeks than the control plants (left, blue). B. Brookshire
  2. Fertilized plants (right, yellow) had longer roots after three weeks than the control plants (left, blue). But the difference was pretty small. B. Brookshire
  3. Fertilized plants (right, yellow) had longer leaves after three weeks than the control plants (left, blue). This difference was larger than the difference seen in the root lengths. B. Brookshire

I started with a hypothesis that fertilized plants will be bigger than those that are not fertilized. Well, the fertilized plants had more leaves, and their leaves were longer. The roots were longer too, though the difference wasn’t very large. Overall, it appears that fertilizer does make radishes grow bigger than they might otherwise.

Of course, every experiment has limitations — things that could have gone better. For example, why did so few of my radishes sprout? I think perhaps if I did it again, I would place my pots in a sunnier spot. I also pulled the plants when they were still small. In another experiment, I would give them more time to grow. After all, we got larger radish greens. It’s possible that with more leaves and more time in the sun — and thus more photosynthesis — we’d end up with larger radishes.

There are lots of other things to try. I could try different “doses” of fertilizer. I could also try different types of radish. Maybe some respond better to fertilizer than others. There’s lots of science that can be done with some dirt and a few seeds.

Materials

  • Miracle Gro ($7.48)
  • Organic potting soil (Bumper Crop, $32)
  • Radish seeds (Cherry Belle, 21-day, $1.95/packet)
  • Seedling pots (.50 each)
  • Measuring cups ($7.46)
  • Nitrile or latex gloves ($4.24)
  • Small digital scale ($11.85)

Power Words

atom: The basic unit of a chemical element. Atoms are made up of a dense nucleus that contains positively charged protons and uncharged neutrons. The nucleus is orbited by a cloud of negatively charged electrons.

ATP: Short for adenosine triphosphate. Cells make this molecule to power almost all of their activities. Cells use oxygen and simple sugars to create this molecule, the main source of their energy. The small structures in cells that carry out this energy-storing process are known as mitochondria. Like a battery, ATP stores a bit of usable energy. Once the cell uses it up, mitochondria must recharge the cell by making more ATP using energy harvested from the cell’s nutrients.

average: (in science) A term for the arithmetic mean, which is the sum of a group of numbers that is then divided by the size of the group.

carbon: The chemical element having the atomic number 6. It is the physical basis of all life on Earth. Carbon exists freely as graphite and diamond. It is an important part of coal, limestone and petroleum, and is capable of self-bonding, chemically, to form an enormous number of chemically, biologically and commercially important molecules. (in climate studies) The term carbon sometimes will be used almost interchangeably with carbon dioxide to connote the potential impacts that some action, product, policy or process may have on long-term atmospheric warming.

carbon dioxide: (or CO2) A colorless, odorless gas produced by all animals when the oxygen they inhale reacts with the carbon-rich foods that they’ve eaten. Carbon dioxide also is released when organic matter burns (including fossil fuels like oil or gas). Carbon dioxide acts as a greenhouse gas, trapping heat in Earth’s atmosphere. Plants convert carbon dioxide into oxygen during photosynthesis, the process they use to make their own food.

cell: The smallest structural and functional unit of an organism. Typically too small to see with the unaided eye, it consists of a watery fluid surrounded by a membrane or wall. Depending on their size, animals are made of anywhere from thousands to trillions of cells. Most organisms, such as yeasts, molds, bacteria and some algae, are composed of only one cell.

chemical: A substance formed from two or more atoms that unite (bond) in a fixed proportion and structure. For example, water is a chemical made when two hydrogen atoms bond to one oxygen atom. Its chemical formula is H2O. Chemical also can be an adjective to describe properties of materials that are the result of various reactions between different compounds.

chemistry: The field of science that deals with the composition, structure and properties of substances and how they interact. Scientists use this knowledge to study unfamiliar substances, to reproduce large quantities of useful substances or to design and create new and useful substances. (about compounds) Chemistry also is used as a term to refer to the recipe of a compound, the way it’s produced or some of its properties. People who work in this field are known as chemists. (in social science) A term for the ability of people to cooperate, get along and enjoy each other’s company.

control: (n.) A part of an experiment where there is no change from normal conditions. The control is essential to scientific experiments. It shows that any new effect is likely due only to the part of the test that a researcher has altered. For example, if scientists were testing different types of fertilizer in a garden, they would want one section of it to remain unfertilized, as the control. Its area would show how plants in this garden grow under normal conditions. And that gives scientists something against which they can compare their experimental data. (v.) To include some unchanged or unaffected conditions in an experiment so their results could later be contrasted with those from where changes had been made.

crop: (in agriculture) A type of plant grown intentionally grown and nurtured by farmers, such as corn, coffee or tomatoes. Or the term could apply to the part of the plant harvested and sold by farmers.

data: Facts and/or statistics collected together for analysis but not necessarily organized in a way that gives them meaning. For digital information (the type stored by computers), those data typically are numbers stored in a binary code, portrayed as strings of zeros and ones.

DNA: (short for deoxyribonucleic acid) A long, double-stranded and spiral-shaped molecule inside most living cells that carries genetic instructions. It is built on a backbone of phosphorus, oxygen, and carbon atoms. In all living things, from plants and animals to microbes, these instructions tell cells which molecules to make.

element: A building block of some larger structure. (in chemistry) Each of more than one hundred substances for which the smallest unit of each is a single atom. Examples include hydrogen, oxygen, carbon, lithium and uranium.

fertilizer: Nitrogen, phosphorus and other plant nutrients added to soil, water or foliage to boost crop growth or to replenish nutrients that were lost earlier as they were used by plant roots or leaves.

function: (in math) A relationship between two or more variables in which one variable (the dependent one) is exactly determined by the value of the other variables.

hypothesis: (v. hypothesize) A proposed explanation for a phenomenon. In science, a hypothesis is an idea that must be rigorously tested before it is accepted or rejected.

mass: A number that shows how much an object resists speeding up and slowing down — basically a measure of how much matter that object is made from.

molecule: An electrically neutral group of atoms that represents the smallest possible amount of a chemical compound. Molecules can be made of single types of atoms or of different types. For example, the oxygen in the air is made of two oxygen atoms (O2), but water is made of two hydrogen atoms and one oxygen atom (H2O).

nitrate: An ion formed by the combination of a nitrogen atom bound to three oxygen atoms. The term is also used as a general name for any of various related compounds formed by the combination of such atoms.

nitrogen: A colorless, odorless and nonreactive gaseous element that forms about 78 percent of Earth's atmosphere. Its scientific symbol is N. Nitrogen is released in the form of nitrogen oxides as fossil fuels burn. It comes in two stable forms. Both have 14 protons in the nucleus. But one has 14 neutrons in that nucleus the other has 15. For that difference, they are known, respectively, as nitrogen-14 and nitrogen-15 (or 14 N and 15 N).

nutrient: A vitamin, mineral, fat, carbohydrate or protein that a plant, animal or other organism requires as part of its food in order to survive.

organic: (in chemistry) An adjective that indicates something is carbon-containing also a term that relates to the basic chemicals that make up living organisms. (in agriculture) Farm products grown without the use of non-natural and potentially toxic chemicals, such as pesticides.

osmosis: The movement of certain molecules within a solution across a membrane. The movement is always from the solution where the concentration of some chemical is higher to the solution where the concentration of that chemical is lower. This movement tends to continue until concentrations on each side of the membrane are the same.

oxygen: A gas that makes up about 21 percent of Earth's atmosphere. All animals and many microorganisms need oxygen to fuel their growth (and metabolism).

p: value (in research and statistics) This is the probability of seeing a difference as big or bigger than the one observed if there is no effect of the variable being tested. Scientists generally conclude that a p value of less than five percent (written 0.05) is statistically significant, or unlikely to occur due to some factor other than the one tested.

phosphate: A chemical containing one atom of phosphorus and four atoms of oxygen. It is a component of bones, hard white tooth enamel, and some minerals such as apatite.

phosphorus: A highly reactive, nonmetallic element occurring naturally in phosphates. Its scientific symbol is P. It is an important part of many chemicals and structures that are found in cells, such as membranes, and DNA.

plastic: Any of a series of materials that are easily deformable or synthetic materials that have been made from polymers (long strings of some building-block molecule) that tend to be lightweight, inexpensive and resistant to degradation.

potassium: A chemical element that occurs as a soft, silver-colored metal. Highly reactive, it burns on contact with air or water with a violet flame. It is found not only in ocean water (including as part of sea salt) but also in many minerals.

probability: A mathematical calculation or assessment (essentially the chance) of how likely something is to occur.

protein: A compound made from one or more long chains of amino acids. Proteins are an essential part of all living organisms. They form the basis of living cells, muscle and tissues they also do the work inside of cells. Among the better-known, stand-alone proteins are the hemoglobin (in blood) and the antibodies (also in blood) that attempt to fight infections. Medicines frequently work by latching onto proteins.

salt: A compound made by combining an acid with a base (in a reaction that also creates water). The ocean contains many different salts — collectively called “sea salt.” Common table salt is a made of sodium and chlorine.

seedling: The initial plant that sprouts leaves and roots after emerging from a seed.

standard deviation: (in statistics) The amount that each a set of data varies from the mean.

statistics: The practice or science of collecting and analyzing numerical data in large quantities and interpreting their meaning. Much of this work involves reducing errors that might be attributable to random variation. A professional who works in this field is called a statistician.

sun: The star at the center of Earth’s solar system. It is about 27,000 light-years from the center of the Milky Way galaxy. Also a term for any sunlike star.

Editor's Note:

This story was updated on December 9, 2020 to correct numbers displayed in the table.

About Bethany Brookshire

Bethany Brookshire was a longtime staff writer at Science News for Students. She has a Ph.D. in physiology and pharmacology and likes to write about neuroscience, biology, climate and more. She thinks Porgs are an invasive species.

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How would one track a mineral nutrient in a plant in order to prove that nutrient has been re mobilized? - Biology

When looking at this table, the Coco sample has extremely low cadmium and lead levels and less than 1/3 maximum allowable chromium (based on European organic rating standards). Theoretically, if you were using low contaminant inorganic fertilizers, the heavy metal count in the end product would be extrmely low. Once again hydroponics is looking very good against organic standards.

Let’s now compare coco substrate to two organic potting mixes.

ORGANIC POTTING MIX 4- 5- 3

ORGANIC POTTING MIX 1-.4-.1

Comparison (mg/kg)

Once again, hydroponics is looking extremely good!

CONTRIBUTING FACTORS TO HM BIOAVAILABILITY/UPTAKE

The Influence of pH on Heavy Metal Uptake

Among the many organic media properties that influence heavy metal uptake by plants, pH plays an important role. 5

In experiments run by Autumn S. Wang et al (2005) cadmium uptake significantly increased in low pH (acidic) high metal concentration soils. The highest rates of Cd uptake occurred at pH 4.8 with a marked decrease in uptake at a higher pH of 5.28, with the lowest uptake rates at pH 6.07. 6 This looks positive due to the optimum pH range for nutrient availability (re cannabis) being 5.8 – 6.0 (with contaminant uptake being minimized within these ranges).

CEC and Heavy Metal Uptake

CEC relates to a soils/substrates ability to attract, retain, and exchange cation elements.

Cation elements are elements with positive electrical charges these being potassium (K+), ammonium (NH4+), magnesium ( Mg++), calcium (Ca++), zinc (Zn+), manganese (Mn++), iron (Fe++), copper (Cu+) and hydrogen (H+). While hydrogen isn’t a nutrient it affects the degree of acidity (pH) of a substrate and, for this reason, is an important consideration.

Some nutrients have negative electrical charges. These are called anions and include nitrate (NO3 N), phosphate, sulfate, borate, and molybdate.

The word “ion” (as in cat –ion and an – ion) simply means a charged particle a positive charge is attracted to a negative charge and vice-versa. This means both positive and negative charged nutrients/elements form a symbiotic relationship and are available for uptake.

High CEC values indicate that a soil or substrate has a greater capacity to hold cations and where there is high CEC there is a large nutrient reserve. Coco substrate has high CEC.

High CEC favors the presence of Cd and other heavy metals in organic media. Put simply, heavy metals bond with organic particles and these often become available for uptake.

Epstein (2003) notes that soils with low CEC such as sands have a much lower binding power as compared to clays with a higher CEC. 7

Due to this, it is imperative that plants grown in high CEC medias are fertilized with the cleanest nutrients/fertilizers possible. This makes product choice (re organic or inorganic nutrients) in formulation imperative. E.g. Low contaminant fertilizers can be manufactured using low contaminant base components. In the case of home formulation, nutrients containing a high degree of analytical, tech and pharmaceutical grade elements can be produced for about the same cost that you are now paying for standard nutrients (formulated with hort grade components) from stores. For instance, phosphate fertilizers can be purchased in food grade and analytical grade calcium nitrate could be used to reduce overall heavy metal contaminants. This is yet another advantage of manufacturing nutrients at home.

Chelators and Heavy Metal Uptake

Well-formulated hydroponic nutrients ensure that there is a high level of nutrient availability in the correct forms and ratios. Nutrition that offers a diverse range of bioavailable elements will prove more effective than nutrition that has less diversity, particularly where trace elements (metals) are concerned. For this reason combinations of organic and synthetic chelates are demonstrated to benefit yields.

The common types of chelates used by most hydro nutrient manufacturers are the synthetic chelates, EDTA (ethylenediaminetetraacetic acid) and to a lesser extent DTPA (Diethylene triamine pentaacetic acid). Chelates such as EDTA and DTPA have a high affinity for e.g. iron and generally form stable complexes with the metal across a pH range from 4 to 7.

DPTA, EDDHA and EDTA are large molecules that aren’t taken up by the plant. Put simply, the chelator leaves the metal ion (micro element) on the roots for uptake and then remains intact in the solution, soil or media. What this means is the chelator remains active in the soil/media and can then chelate heavy metals making them more bioavailable for uptake (I.e. the chelator bonds with heavy metals such as cadmium and delivers them to the roots of the plant for uptake).

All manufacturers use synthetic chelates in formulation. For instance, typically Iron will be supplied as Fe EDTA, with some manufacturers using Fe DTPA and/or Fe EDDHA. The bottom line – chelates aid micro element uptake – the downside is that heavy metals such as cadmium also become more bioavailable to cannabis when they are chelated. 1

Some manufacturers are now identifying with this issue. For instance, Yarra (an agricultural fertilizer manufacturer) is currently working on producing biodegradeable chelates for use in agriculture.

Other than this, organic chelators (e.g. amino acids, fulvic acid), unlike the synthetic chelators are absorbed into the plant and not left lying around to chelate heavy metals. This makes organic chelators ideal for reducing heavy metal uptake, while still ensuring optimum yields.

Organic Chelators

The synthetic chelate EDTA does not penetrate the root. The chelate leaves the metal on the root surface before the root absorbs it. The synthetic chelator is then left in the nutrient, soil or media. Upon entering the plant the metal will immediately become chelated again by organic acids such as citric acids, malonic acid, tartaric acid (tartrate), and some amino acids (e.g. glycine) which occur naturally within the plant. This chelation process will then enable the nutrients to move freely inside the plant to areas where they are most needed.. Which brings us to our next point. Micro Proteinates – otherwise known as ‘glycinates’ or ‘proteinates’ – which can be purchased as zinc, boron, calcium, magnesium, iron, manganese and copper.

Amino Proteinates/Glycinates (Organic Chelates)

Amino acids are the “building blocks” of protein without which the formation of any living tissue is impossible.

Amino acids such as glycine are organic chelating agents that are naturally occurring in plants. Glycine is the simplest amino acid with a molecular weight of 75. Chelates of glycine with cations such as iron, zinc, and copper have been extensively studied. For instance, research conducted in USSR established that glycinates greatly stimulate the growth of plants. The results concluded that zinc glycinate (zinc glycine chelate) increased the total, stem, root, and foliage weights by 194, 215, 254 and 147%, respectively. Respective effects of manganese glycinate (manganese glycine chelate) were 79, 108, 110, and 15%.

Glycinates (proteinates) are organic chelates and unlike the synthetic chelates are absorbed along with the metal into the plant. This offers distinct advantages over synthetic chelators.

Glycinates contain 2 moles of ligand (glycine) and one mole of metal. The plant recognises this molecule as a protein like nitrogen, allowing it to travel to the growing points such as flowers, fruit and berries where is it required.

Micronutrients in proteinate/glycinate form have a very stable structure. They can be easily absorbed through the roots and directly join the biochemical processes in the plant.

Research has demonstrated:

1. Glycinates increase the availability of micronutrients compared to common synthetic chelates (e.g. EDTA, DTPA).

2. Crops tend to produce higher yields where glycinates are used.

For this reason, the use of at least some glycinates in high-end med formulation is recommended.

1. B. Kos and D.Lestan Soilwashing of Pb, Zn and Cd using biodegradable chelator and permeable barriers and induced phytoextraction by Cannabis sativa

Biological Microorganisms and Heavy Metal (HM) Uptake

Microbial populations are known to affect heavy metal mobility and availability to the plant through release of chelating agents, acidification, phosphate solubilization and redox changes and, therefore, microorganisms (in some cases) have the potential to enhance HM uptake.

Research demonstrates that microorganisms such as Bacillus subtillus 8 , mycorrhizae (AM, AMF, VAM) 9 and Trichoderma harzianum (Trichoderma SPP) 10 play a role in heavy metal uptake rates. Given these micros are used by many med growers (and others) as biological innoculants and plant growth enhancers/stimulants this becomes an important area of consideration.

Benefits of Microbes: In Brief

When researching the literature on microbes and heavy metal uptake, it became apparent that the interaction of microbes and heavy metal uptake from soils depended on several key factors. These were:

  • Most significantly – Heavy metal concentration in soils, substrates and fertilizers (compost etc) is the dominant factor in HM plant tissue contamination
  • Heavy metal type, relative to microbe species
  • Plant species
  • Nutrient levels and their effect on microbe colonization (biomass)

Beneficial microbes have been demonstrated to enhance root growth, plant growth, and yields. Other than this, microbes play an important role as biofungicides, crop protectants and plant immune stimulants. For this reason the role of beneficial microbes in sustainable agriculture and bio agriculture cannot be underestimated.

Beneficial Microbes can aid nutrient uptake and act as, among other things, (put simply) chelators of metal ions (I.e. make elements such as Fe, Cu, Mn, Zn more available for uptake and translocation).

Optimum chelation will also increase the potential for heavy metal uptake. I.e. In chelating metals such as Fe, Cu, Mn, Zn you are also chelating heavy metal ions such as Cd, Pb and Hg.

Arbuscular Mycorrhiza Fungi (mycorrhizae referred to as AM, VAM or AMF)

Mycorrhiza (AM) improves nutrient transfer from the soil to the roots of the host plant. Numerous trials have demonstrated increased biomass, yield weights, and root growth in AM colonized crops.

Weissenhorn et al states that under optimized conditions of normal agricultural practices AM may increase plant heavy metal (HM) absorption. 11 However, Voros et al (1998) notes that many of the studies on heavy metal uptake in AM colonized soils are contradictory. 12

Research on Heavy Metal Uptake and AM

Citterio Et al (2004), in research with Cannabis Sativa grown in HM contaminated soils populated with Glomus mosseae, discovered that Cd uptake and translocation was increased in heavily contaminated soils, while it Cd uptake was not influenced in partially contaminated soils.

“… plants grown in artificially contaminated soil accumulated most metal in root organ. In this soil, mycorrhization significantly enhanced the translocation of all the three metals from root to shoot.” 13

However, research on tobacco by M. Janoušková et al (2004) found that Cd uptake was reduced when AM was present in soils.

“AM decreased the Cd uptake of the tobacco plants per unit of shoot biomass in both experiments and decreased the Cd accumulation in the shoots of the transgenic tobacco relatively to the non-transgenic tobacco. “ 14

Further research supports these findings, T. Takács et al (2002) note that AM reduced Cd uptake in ryegrass 15 : El-Kherbawy et al (1988) demonstrated that AM “significantly” reduced heavy metal uptake in experiments with Alfalfa (Medicago sativa L.). 16

Seemingly, VAM reduce HM uptake in enriched soils and substrates due to the hyphal complexes of mycorrhizae providing absorptive surfaces within the cortical cells of the host roots, thereby excluding metals from the shoot. 17

Mycorrhizae Viability in Hydroponic Settings

The key to AM viability in hydroponics is that mycorrhizae will not colonize efficiently in high phosphorous environments (e.g. hydroponics, where traditional nutrients are used). Put simply, AM fix phosphorous and where high phosphorous already exists in fertilizers, soils or substrates, AM colonization and translocation is demonstrated to be ineffective. 18 What this means (in layman’s terms) is that nutrients must be formulated specifically with low phosphorous (P) levels to cater for mycorrhizae colonization/viability.

AM friendly formulas should optimally deliver no more than 10ppm of P (as dilute feed solution) in order to achieve efficient AM colonization. E.g.

Element ppm (nutrient values on delivery)

Bacillus Subtillus

Where Arbuscular mycorrhiza fungi seemingly have the potential to reduce HM uptake, research demonstrates that Bacillus subtillus and Bacillus pumilus bacterial strains (16S rRNA gene sequence strains) increase HM uptake. 19

Research by Hawkins et al with Z. mays (corn) and S. bicolor (sorghum/maize) demonstrated that B.subtillus and B. pumilus play an important role in increasing metal availability in soil, enhancing Cr, Pb, Zn and Cu uptake. 20

Trichoderma Harzianum

Trichoderma spp (e.g. T. harzianum, T. viride, T. koningil, T. hamatum) facilitate robust root growth, increase plant growth, increases nutrient uptake and fertilizer utilization, and enhances plant greenness, which may result in higher photosynthetic rates. Trichoderma spp also have been known for a very long time to have the ability to control plant pathogenic fungi.

Trichoderma species are used to produce cellulases. They are particularly effective as antagonists of the growth of other fungi, many of them plant pathogens, with the result that trichoderma species are important biocontrol agents.

J. S. Chauhan et al (2009) found that a combination of Pseudomonas fluorescens and Trichoderma harzianum enhanced the uptake of Zn and Cd in Indian mustard (Brassica juncea) from soils containing three different concentrations of Zn (300, 600, 900 mg/kg) and Cd (5, 10 and 15 mg/kg). 21

1. J.C Lgwe, E.C.Nwokennaya1 and A.A. Abia (2000) The role of pH in heavy metal detoxification by bioabsorption from aqueous solutions containing chelating agents

2. New Zealand Biological Producers Council. Organic soil management in New Zealand.

3 and 4. Heavy Metals and Organic Compounds from Wastes Used as Organic Fertilizers: Compost Quality Definition Legislation Standards. Technical Office for Agriculture (Austria)

5. Autumn S. Wang1,5, J. Scott Angle1, Rufus L. Chaney2, Thierry A. Delorme3

& Roger D. Reeves (2005) Soil pH effects on uptake of Cd and Zn by Thlaspi caerulescens.

6. Autumn S. Wang et al (2005)

7. Epstein, E (2003) Land Application of Sewage Sludge and Biosolids.

8. R. A. Abou-Shanab , K. Ghanem, N. Ghanem and A. Al-Kolaibe (2007) The role of bacteria on heavy-metal extraction and uptake by plants growing on multi-metal-contaminated soils

9. Weissenhorn, C. Leyval, G. Belgy and J. Berthelin (1995) Arbuscular mycorrhizal contribution to heavy metal uptake by maize (Zea mays L.) in pot culture with contaminated soil

10. ADAMS P. DE-LEIJ F. A. A. M. LYNCH J. M. (2009) Trichoderma harzianum rifai 1295-22 mediates growth promotion of cracl willow (Salix fragilis) saplings in both clean and metal-contaminated soil

11. I. Weissenhorn, C. Leyval, G. Belgy and J. Berthelin. (2004) Arbuscular mycorrhizal contribution to heavy metal uptake by maize (Zea mays L.) in pot culture with contaminated soil

12. T. Takacs, B. Biro and I. Voros (2002) Arbuscular mycorrhizal effect on heavy metal uptake in ryegrass (Lolium perenne L.) in pot cultured with polluted soils.

13. J. Citterio, N. Prato, P. Fumigalli, R. Aina, N. Massa, A. Santagostino, S. Sgorbati, G. Berta (2004) The arbuscular mycorrhizal fungus Glomus mosseae induces growth and metal accumulation changes in Cannabis sativa L.

14. M. Janoušková , D. Pavlíková T. Macek, and M. Vosátka (2004) Influence of arbuscular mycorrhiza on the growth and cadmium uptake of tobacco with inserted metallothionein gene

15. T. Takacs, B. Biro and I. Voros (2002) Arbuscular mycorrhizal effect on heavy metal uptake in ryegrass (Lolium perenne L.) in pot cultured with polluted soils.

16. M. El-Kherbawy, J,S. Angle, A. Heggo, and RL. Chaney (1988) Soil pH, rhizobia, and vesicular-arbuscular mycorrhizae inoculation effects on Growth and heavy metal uptake of alfalfa (Medicao sativa L)

17. Bradley R, Burr AJ, Read DJ (1982) The biology of mycorrhiza in the Ericaceae VIII. The role of mycorrhiza infection in heavy metal resistance. New Phytol 91:197-209

18. H.-J. Hawkins and E. George (2004) Hydroponic culture of the mycorrhizal fungus Glomus mosseae with Linum usitatissimum L., Sorghum bicolor L. and Triticum aestivum L.

19. R. A. Abou-Shanab , K. Ghanem, N. Ghanem and A. Al-Kolaibe: The role of bacteria on heavy-metal extraction and uptake by plants growing on multi-metal-contaminated soils

20. R. A. Abou-Shanab , K. Ghanem, N. Ghanem and A. Al-Kolaibe: The role of bacteria on heavy-metal extraction and uptake by plants growing on multi-metal-contaminated soils

21. J. S. Chauhan J. P. N. Rai (2009) Phytoextraction of soil cadmium and zinc by microbes-inoculated Indian mustard (Brassica juncea)

HIGH THC CANNABIS SPECIFIC RESEARCH

In 2001, the NSW Government (Australia) issued a permit to Nimbin local Andrew Kavasilas to allow a limited amount of high THC cannabis to be grown for research and analytical purposes. The research was undertaken with the assistance of the Centre for Phytochemistry (and its commercial arm Australian Phytochemicals Ltd) at the Southern Cross University in Lismore NSW.

Kavasilas tested three strains of outdoor organically cultivated cannabis and three variations of indoor hydroponic cannabis (commercial NSW, commercial SA and “home grown”) for heavy metal contaminants. His findings suggest:

  1. Genetics seemingly play a role in heavy metal uptake and translocation rates of at least some heavy metals *1
  2. Nutrition (contaminant levels in soils and fertilizers) plays by far the most significant role in heavy metal contamination (i.e. the more heavy metals in solution/fertilizers and soils the higher the potential rate of contamination in plant tissue)
  3. That based on the growing procedures (trial methodology) organic nutrition/fertilizers resulted in less heavy metal contamination than inorganic hydro fertilzers although this finding is largely inconclusive due to two other indoor samples (commercially grown indoor hydro cannabis from SA and NSW) yielding equivalent levels of heavy metal contaminants to three outdoor samples with only one sample – Kavasilas’ own home grown (hydro) indoors – yielding far higher levels of heavy metal contaminants than all other (5) samples. * 2 Further, Kavasilas research is in contrast to more comprehensive testing conducted by Advanced Nutrients in 2002-2003.

*1 Kavasilas grew Afghan, Durban and Skunk x Northern Lights outdoors. Leaf tissue analysis found 1.10mg/kg of lead in Durban with <0.1 in both the Afghan and Northern Lights x Skunk. Chromium was 4.10 (Durban), 4.30 (Afghan) and 2.40 (Skunk x Northern Lights). If no other variables (i.e. nutrition, heavy metal content in soil, and feed rates) were influencing factors this indicates that various strains of cannabis may uptake, at least, some heavy metals at differing rates.

*2 Cadmium in all samples (3 x organic and 3 x indoor hydro) was <0.1mg/kg with the exception of Kavasilas’ own “home grown” sample at 3.70mg/kg. In all instances Kavasilas’ “home grown” indoor cannabis tested significantly higher for contaminants than two separate samples of indoor cannabis and three samples of outdoor (5 samples total). The two samples of “commercial” indoor cannabis tested at the same rates as outdoor cannabis with the exception of chromium where both indoor samples tested lower (by average) than outdoor samples. Further research is recommended.

See findings below. Extracted from Medical Uses of Cannabis by Andrew Kavasilas (2003)

Advanced Nutrients Research (tissue analysis) 2002 – 2003

COMMENTS: Total Metals, Samples Digested 22 Nov 02

METHODS: Standard Methods for the Examination of Water and Wastewater

White Rhino at 56 days had:

Discussion

When comparing the Advanced Nutrients data to that of Andrew Kavasilas’ data the results are for the most part consistent bar for Kavasilas’ own homegrown hydro which inexplicitly has higher levels of HM contamination than all other hydroponically grown produce. This raises concerns as to the accuracy of Kavasilas’ data in one instance.

What is apparent based on all tests is that low levels of cadmium, low levels of lead, low levels of chromium and low levels of arsenic are present in both hydroponically grown and organically grown cannabis. Aluminum, a non heavy metal element, but potentially toxic nevertheless, may also pose a problem and more research into this area is needed.

Further, comparing the two sets of data suggests that higher HM counts are present in organically grown produce than are present in hydroponically grown produce.

Recommendations for minimizing cadmium and other heavy metal exposure

  • Cannabis/medicine should be grown using hydroponic and/or organic growing methodologies using fertilizers (and soils re organics) that are low in heavy metal contaminants
  • Cannabis/medicine should ideally be grown using non-organic fertilizers formulated with laboratory, food, and/or analytical and pharmaceutical grade fertilizers to minimize HM contamination.
  • Where organic fertilizers are used, products should be tested for Cd and other HM levels or a guaranteed analysis that includes heavy metal content should be obtained.
  • Med dispensaries should collate a list of various organic and inorganic fertilizers (brands) and their heavy metal content – this information should then be made available to med growers. Lab tests for Cd and other contaminants should be conducted independently
  • Immunosuppressed individuals should eat or vaporize their medicine to minimize Cd and other contaminant absorption/exposure

Author’s note 1: Over the years I have seen various “hydro” manufacturers make claims as to the use of analytical or pharmaceutical grade elements in their products. After analyzing some of these products (and investigating through other means) it became clear that while they may (and I stress “may”) be incorporating some low contaminant fertilizers in formulation their use – if any – was extremely low, with the bulk of the formula being manufactured from standard horticultural grade base components. For instance, having looked at formulas from one company who claims to use pharmaceutical grade elements in formulation it became clear that the use of pharmaceutical grade components was minimal/minute (0.27% of the total mineral weight used in production). Other than this, after running lab analysis on another brand that claims to use analytical or food grade elements it became clear that if they were using analytical grade, the contaminant levels looked very much like standard fertilizers produced from hort grade products. After vieweing some of their promotional material (a video) where Yarra Fertilizers (hort grade products) appeared to be used in manufacture the sitution became somewhat dubious. I.e. it looked like this particular manufacturer was engaging in suspect marketing.

Author’s note 2: Due to the higher purchase cost of analytical, food and pharmaceutical grade elements one must be wary of claims as to blends containing these (stated) elements (this may change and I will keep you advised/updated). Put simply, the hydro industry is a cost driven market and manufacturers simply can’t formulate competitively priced products using expensive base components. By way of example, one manufacturer claims to use British Pharmaceutical grade elements in production. As a guestimate (based on component purchase prices, wholesale and retail markups) a product entirely formulated with these elements would need to retail at $400.00USD plus for a 5L set. This is yet another advantage of formulating yourself. I.e. for about the same cost that you pay for a hort grade product in stores you will be able to formulate using analytical, food grade and pharmaceutical grade components.

1. Department for Health and Human Services, Agency for Toxic Substances and Disease

2. The Ministry of Agriculture and Forestry, NZ

3. Jarup, L. (1998) Health effects of cadmium exposure—a review of the literature and a risk estimate

4. Department for Health and Human Services, Agency for Toxic Substances and Disease Registry, 2008

5. P, Linger et al (2001) Industrial hemp (Cannabis sativa L.) growing on heavy metal

contaminated soil: fibre quality and phytoremediation potential

6. Marth, E et al (2000) Influence of cadmium on the immune system. Description of

stimulating reactions. Central European journal of public health

7. Food and Agriculture Organization of the United Nations. http://www.fao.org

8. Burger J et al (2001) Kelp as a bioindicator: Does it matter which part of 5 M long plant is used for metal analysis

9. Boening D et al (1999) Ecological effects, transport, and fate of mercury: a general review

10. Curtis L. and Smith. B (2002) Heavy Metal in Fertilizers: Considerations for Setting Regulations In Oregon Department of Environmental and Molecular Toxicology, Oregon State University Corvallis, Oregon

11. Gulz, P (2003) Arsenic Uptake of Common Crop Plants from Contaminated Soils and Interaction with Phosphate. University of Munich

12. Andrew Kavasilas (2003) Medical Uses of Cannabis, ISBN 0-9751806-0-6

13. Jones, Clement and Hopper (1973) Lead uptake from solution by perennial ryegrass and its transport from roots to shoots.

14. Kumar et al (1995) Phytoextraction: The use of plants to remove heavy metals from soils.

15. Kos, B. Gremen, H. Lestan, D (2003) Phytoextraction of lead, zinc and cadmium from soil by selected plants

16. Andrew Kavasilas (2003) Medical Uses of Cannabis

17. Golovatyj SE, (1999) Effect of levels of chromium content in a soil on its distribution in organs of corn plants. Soil Res Fert 197– 204.

18. Prato N et al (2003) Cannabis sativa for heavy metal contaminated soil restoration

19. European Commission (2005) Communication from the Commission to the Council and the European Parliament on Community Strategy Concerning Mercury

20 B. Z. Siegel, Lindley Garnier and S. M. Siegel (1988) Mercury in Marijuana

21. Batáriová A et al (2005) Blood and urine levels of Pb, Cd and Hg in the general population of the Czech Republic and proposed reference values

22. B. Z. Siegel, Lindley Garnier and S. M. Siegel (1988) Mercury in Marijuana

23. Patra M. and Sharma A (2000) Mercury Toxicity In Plants

24. United States Environmental Protection Agency (Dec 1997) Mercury study report to congress

25. B. Z. Siegel, Lindley Garnier and S. M. Siegel (1988) Mercury in Marijuana

27. Andrew Kavasilas (2003) Medical Uses of Cannabis, ISBN 0-9751806-0-6

Other Medical Marijuana Contaminants

Mycotoxins/Aflatoxins

Spores of fungi/moulds are common in cannabis. At least 88 species of fungi attack Cannabis and more are being discovered every year (McPartland & Hughes 1994, McPartland & Cubeta 1996). Research has demonstrated that fungi spores survive in smoke inhaled from marijuana cigarettes. Most fungi are plant pathogens and their ingestion will typically not harm healthy humans however, some fungi cause harm by producing secondary toxins.

Studies have reported levels of biological contaminants in cannabis, which include Aspergillus fungus and bacteria, potentially leading to fulminant pneumonia, especially among the immunosuppressed.

A.flavus (Aspergillus flavus), is a common mould that is found in the environment and produces aflatoxins. Aflatoxins are naturally occurring mycotoxins and are among the most carcinogenic substances known they are just under 2000 times more toxic than even the most toxic pesticides. Aflatoxins survive combustion and, therefore, pose a risk to medial marijuana consumers.

A.flavus has a worldwide distribution. Although it is universally found in air, soil, dust and water, higher fungal burdens have been noted particularly in contaminated peanuts, corn, grains and decaying organic matter. Disease prevalence is highly variable with different institutions reporting different species of Aspergillus as the predominant pathogen.

The primary mode of transmission to humans is by inhalation. Aspergillus spores are released in the air and may remain airborne for prolonged periods. As a result, spores are ubiquitously found in air and contaminate anything they in contact with, including plants. There is increasing concern about contaminated food, environmental and occupational exposure to fungal spores of different species, especially to the aflatoxin-producing strains of A. flavus, in different parts of the world. Higher frequency of pulmonary function impairment and allergic respiratory diseases including asthma has been reported in farmers around the world. In addition to inhalation, a secondary route of transmission has been reported via contact with skin or wound (trauma and postoperative), contamination of intravenous solutions, wound dressings and marijuana inhalation.

About 185 different species of Aspergillus have been identified, of which 20 are documented to cause human disease. Aspergillus spores, upon inhalation, can lead to colonisation, allergic manifestations or invasive infection depending on the host’s immune system. Invasive aspergillosis is rare in people who have a good immune system but contributes to significant morbidity and mortality in immunosuppressed patients. “Aspergillosis” refers to several forms of disease caused by a fungus in the genus. The majority (approximately 80%) of invasive Aspergillus infections are caused by Aspergillus fumigatus. The second most frequent (approximately 15–20%) pathogenic species is Aspergillus flavus and to a lesser extent, Aspergillus niger and Aspergillus terreus. Aspergillus flavus has emerged as a predominant pathogen in patients with fungal sinusitis and fungal keratitis in several institutions worldwide.

Conidium (fungal spores) of the most commonly involved pathogenic Aspergillus species (spp.) are relatively small, with sizes ranging from 2 to 5 micron. Due to their small size, conidium will become deposited deep into the lung after inhalation. In most individuals, inhaled conidia will be cleared, without affecting the individuals health. Immunocompromised patients, however, are extremely susceptible to local invasion of respiratory tissues by deposited conidium, resulting in invasive aspergillosis. Aspergillus. Aspergillosis fungal infections can occur in the ear canal, eyes, nose, sinus cavities, and lungs. In some individuals, the infection can even invade bone and the membranes that enclose the brain and spinal cord. Most cases of invasive aspergillosis present with pneumonia. Therefore, it has been hypothesized that the inhalation of airborne Aspergillus conidium is a direct cause of pulmonary infection in immunocompromised patients.

While research is limited surrounding aspergillosis-related pulmonary illness, resulting from marijuana use by immunecompromised AIDS sufferers high fatality rates are shown in relevant medical research. Invasive aspergillosis occurs in advanced AIDS and most commonly affects the lungs, although brain involvement has also been frequently reported.

Other high-risk groups include bone marrow transplant recipients and patients with central nervous system or disseminated aspergillosis.

In one case a 34-year-old man presented with pulmonary aspergillosis 75 days after he had undergone a marrow transplant for chronic myelogenous leukemia. The patient had smoked marijuana heavily for several weeks prior to admission. Cultures of the marijuana revealed Aspergillus fumigatus with morphology and growth characteristics identical to the organism grown from open lung biopsy specimen. Despite aggressive antifungal therapy, the patient died.

In another case a 46-year-old patient with acute myeloid leukemia (AML) whose disease manifested as fever, chills and dry cough was admitted to hospital. Despite broad antibiotic coverage he remained acutely ill with spiking fever, shaking chills, and hypoxemia. A thorough investigation revealed that before becoming acutely ill the patient smoked daily tobacco mixed with marijuana from a “hookah bottle”. While waiting for tobacco and “hookah water” cultures, doctors started antifungal therapy. Resolution of fever and hypoxemia ensued after 72 hours. Tobacco cultures yielded heavy growth of Aspergillus species, suggesting that habitual smoking of Aspergillus-infested tobacco and marijuana caused airway colonization with Aspergillus. Leukemia rendered the patient immunocompromised, and allowed Aspergillus to infest the lung parenchyma with early occurrence of invasive pulmonary aspergillosis.

Moody et al. (1982) have evaluated waterpipes for smoking Aspergillus- contaminated marijuana. They found only a 15% reduction in transmission of fungal spores.

In yet other cases, Chusid et al. blamed Aspergillus fumigatus for causing near-fatal pneumonitis in a 17-year-old. They noted that the patient had buried his marijuana underground for “aging”, creating an ideal environment for microbiological contamination. Similarly, Llamas et al recovered Aspergillus fumigatus from marijuana owned by a patient suffering bronchopulminory aspergillosis, while Shwartz scraped Aspergillus flavus from the sinuses of a marijuana smoker who suffered severe headaches.

Aspergillus flavus contamination has been identified in Dutch coffee shop products.

Additionally, in research conducted by Steven L. Kagen et al (1983) the authors note:

“The possible role of marijuana (MJ) in inducing sensitization to Aspergillus organisms was studied in 28 MJ smokers by evaluating their clinical status and immune responses to microorganisms isolated from MJ. The spectrum of illnesses included one patient with systemic aspergillosis and seven patients with a history of bronchospasm after the smoking of MJ. Twenty-one smokers were asymptomatic. Fungi were identified in 13 of 14 MJ samples and included Aspergillus fumigatus, A. flavus, A. niger, Mucor, Penicillium, and thermophilic actinomycetes. Precipitins to Aspergillus antigens were found in 13 of 23 smokers and in one of 10 controls, while significant blastogenesis to Aspergillus was demonstrated in only three of 23 MJ smokers. When samples were smoked into an Andersen air sampler, A. fumigatus passed easily through contaminated MJ cigarettes. Thus the use of MJ assumes the risks of both fungal exposure and infection, as well as the possible induction of a variety of immunologic lung disorders.”

A study by Verweij et al (2000) showed that samples of both tobacco and marijuana were heavily contaminated with filamentous fungi including A. fumigatus.

And in research conducted by M. Halt (1998), the level of toxigenic moulds and mycotoxins were analyzed in 62 samples of medicinal plant material. The most predominant fungi detected were: Aspergillus, Penicillium, Mucor, Rhizopus, Absidia, Alternaria, Cladosporium and Trichoderma. Aspergillus flavus, was present in 11 or 18% of the 62 medicinal plant samples. The medicinal plant samples, contaminated with A. flavus were also analyzed for the mycotoxins aflatoxin, ochratoxin and zearalenone ochratoxin was found in one of the 7 samples analyzed. The study suggests that medicinal plant material, if stored improperly, allowed for mould growth after harvest.

Microbiological Toxins and Medical Marijuana Screening

The Dutch Medical Marijuana program was legalized in 2003. Growing, processing and packaging of the plant material are performed according to pharmaceutical standards and are supervised by the official Office of Medicinal Cannabis (OMC).

The quality is guaranteed through regular testing by certified laboratories. Under Dutch regulations medical marijuana should contain no heavy metals, pesticides, or fungus contaminants.

However, in the Netherlands a tolerated illicit cannabis market exists in the form of ‘coffeeshops’, which offers a wide variety of cannabis to the general public as well as to medicinal users of cannabis. Since cannabis has been available in the pharmacies, many patients have started to compare the price and quality of OMC and coffeeshop cannabis. As a result, the public debate on the success and necessity of the OMC program has been based more on personal experiences, rather than scientific data.
The general opinion of consumers is that OMC cannabis is more expensive, without any clear difference in the quality.
In 2005, a study was conducted in order to show any differences in quality that might exist between the official and illicit sources of cannabis for medicinal use. Cannabis samples obtained from randomly selected coffeeshops were compared to medicinal grade cannabis obtained from the OMC in a variety of validated tests. Many coffeeshop samples were found to contain less weight than expected, and all were contaminated with bacteria and fungi. No obvious differences were found in either cannabinoid- or water-content of the samples. The obtained results show that medicinal cannabis offered through the pharmacies is more reliable and safer for the health of medical users of cannabis. 1

Similarly, Canada, like Holland, has a legalized medical marijuana program and thousands of Canadians are federally licensed to possess and use medical marijuana through Health Canada. In Canada, medical marijuana is tested not just for active ingredients such as THC, but for mold, fungus, pathogens — including bacteria — and metals, such as lead, cadmium, mercury and arsenic. Further, government regulated medical marijuana is gamma-irradiated for safety purposes to ensure no harmful mould spores are present.

A similar situation has occurred in the U.S. where, although, the U.S. medical marijuana industry is not legalized at a federal level, the industry itself, in some cases, has established standards to ensure that medical marijuana sold to consumers is not contaminated by aflatoxins.

For instance, Steep Hill Cannabis Analysis Laboratory notes, that while 85 percent of the marijuana tested at Steep Hill Lab has shown traces of mould, only 3 percent of those samples have been deemed unsafe under general guidelines for herbal products. 2

Harborside Health Center, Oakland’s largest med dispensary, note on their website that approximately 2% – 4% of cannabis they test is found to show positives for pathogenic moulds at levels where the produce is rejected. 3

However, these numbers appear low when compared to those of the Werc Shop which had a “Gold level of microbiological testing” and is modeled after USP ( U.S. Pharmacopeial Convention) designations levels for dried botanicals. USP Reference Standards are closely tied with the documentary standards published in the USP–NF, Food Chemicals Codex, and Dietary Supplements Compendium. Materials based directly on official monographs in the USP–NF—whose standards and procedures are enforceable by the U.S. Food and Drug Administration—are recognized for use in official standards in the United States, and their use is effective in demonstrating compliance with statutory requirements. Under USP testing procedures the WercShop sees approximately 30% failure rates in the microbiological classification.

1) Arno Hazekamp (2006) An evaluation of the quality of medical grade cannabis in the Netherlands

Prevention of Aflatoxins/Mycotoxins

Growroom Practices

Aspergillus spores drift on air currents, dispersing themselves both short and long distances depending on environmental conditions. When the spores come in contact with a solid or liquid surface, they are deposited and if conditions of moisture are right, they germinate (Kanaani etal., 2008). In all cases fungi consume organic material wherever humidity and temperature are adequate. For Aflatoxins this is a warm and moist/damp environment where fungi proliferate and mycotoxin levels can become high. Mycotoxins resist decomposition, so they can easily remain in cannabis product, post harvest. To minimize risk of aflatoxin infection ensure that air humidity (RH) is between 45-55% and that the growroom has adequate airflow. Use an exhaust fan during the night cycle as well as the day cycle because humidity climbs quickly during the night cycle in rooms that are not vented during this period.

Aspergillis grow abundantly on decaying vegetation where it has been found in large numbers in mouldy hay, organic compost piles, leaf litter and the like. Most species are adapted for the degradation of complex plant polymers. For this reason, the growroom should be kept clean of decaying organic matter at all times.

Fungal growth and aflatoxin contamination are the consequence of interactions among the fungus, the host and the environment. The appropriate combination of these factors determine the infestation and colonization of the substrate, and the type and amount of aflatoxin produced. However, a suitable substrate is required for fungal growth and subsequent toxin production, although the precise factor(s) that initiates toxin formation is not well understood. Water stress, high-temperature stress, and insect damage of the host plant are major determinig factors in mold infestation and toxin production. Similarly, specific crop growth stages, poor fertility, and high crop densities have been associated with increased mold growth and toxin production. Aflatoxin formation is also affected by associated growth of other molds or microbes . For example, preharvest aflatoxin contamination of peanuts and corn is favored by high temperatures, prolonged drought conditions, and high insect activity while postharvest production of aflatoxins on corn and peanuts is favored by warm temperatures and high humidity.

As with people, aflatoxins can more easily colonize a sick plant (host) than a healthy plant. For this reason, optimal growroom conditions should be maintained (temperature, light, airflow, CO2 levels, RH etc) which, in turn, promotes optimal growth/yields and plant health. (Read more on optimum growroom parameters here)

Cleanliness is next to godliness! Thoroughly clean and bleach the growroom between and during crop cycles. Ensure any rotting vegetative matter is removed. Clean up water spills immediately as they can contribute to an increase in air humidity (RH).

Filter Inlet Air Using HEPA Filters

Because mycotoxins/aflatoxins are a common mould found in the outside environment, HEPA (High-Efficiency Particulate Arresting) filters should ideally be placed on inlet fans to keep bacteria, pests, and fungus commonly found in outdoor environments from the growroom. HEPA filters are critical in the prevention of the spread of airborne bacterial and viral organisms and, therefore, infection in hosts. Research has shown that where HEPA filters are employed in hospital settings, aflatoxin counts are greatly reduced – if not eliminated totally. Some of the best-rated HEPA units have an efficiency rating of 99.995%, which assures a very high level of protection against airborne disease transmission.

Water Treatment

Aflatoxins/mycotoxins have been detected in water storage tanks and in chlorine treated mains water supplies. 1 In one study it was shown that Norwegian municipal drinking water may be an important contributor to the transmission of a wide variety of mold species to water consumers 2

It is important to note that RO water filtration does not remove fungal pathogens and, in fact, may increase their incidence due to biomass build up in the filters. For this reason, pharmaceutical grade marijuana production should incorporate treatment of water to remove aflatoxin/mycotoxin contaminates. There is a very simple way to do this.

Use of Beneficial Bacteria and Fungi is Solution (B.subtilus and Trichoderma spp.)

Bacillus subtilis produces peptidolipid compounds of the iturin group that have been shown to have antifungal properties. In one study, the activity of iturin A, produced by B. subtilis strain B-3, was tested. Paper disks impregnated with various concentrations of iturin A were placed on agar plates seeded with conidia of toxigenic species of Fusarium, Gerlacia, Penicillium or Aspergillus. Most isolates were inhibited at where even low levels of B. subtilus were present. italicum, P. vindicatum, A. ochraceus and A. versicolor were most strongly inhibited. 3

Calistru and McLean reported that two isolates of T. harzianum and T. viride were capable of inhibiting the growth of A. flavus. 4 The primary mechanism of antagonism of Trichoderma is microparasitism. Trichoderma also produce volatile and non-volatile antibiotics to suppress target pathogens.

An added benefit to using beneficial bacteria and fungi in solution is they inhibit other pathogenic moulds such as pythium and fusarium – protecting the plants against all manner of disease (read more about beneficial bacteria and fungi in hydroponics here)

1) Identification, Significance and Control of Fungi in Water Distribution Systems. Joan Kelley, Russell Paterson, Graham Kinsey: International Mycological Institute, Bakeham Lane, Egham,

2) Sybren de Hoog and Ida Skaar Gunhild Hageskal, Ann Kristin Knutsen, Peter Gaustad, G (2006) Diversity and Significance of Mold Species in Norwegian Drinking Water

3) M. A. Klich, A. R. Lax and J. M. Bland (1991) Inhibition of some mycotoxigenic fungi by iturin A, a peptidolipid produced by Bacillus subtilis

4) C. Calistru, M McLean and P. Berjak (1997) In vitro studies of the potential for biological control of Asperellis flavus and Fusarium moniliforme by Trichoderma species.

Drying, Storage, and Handling

Fungi and bacteria can inhabit plant material after it has been harvested. For instance, Aspergillus is a commonly occurring fungi in the environment and once a crop has been harvested and there are no residual fungicide then it is still susceptible to Aspergillus.
Drying
The infestation by opportunistic fungi cannot occur in plant material below 15% moisture content (MC). Properly dried marijuana contains approximately 10% MC (material below 10% MC becomes excessively brittle). Consumers should prevent marijuana from reabsorbing moisture above 15% MC.

Dried cannabis should be stored in vacuum-sealed bags and/or sellable quantities (grams, eighths etc) stored/supplied in snap lock bags that can be air released and resealed immediately after use.

Med dispensaries and users should ensure that hygienic practices in handling cannabis products are adhered to.

Medical Best Practice

Carefully cultivated and harvested marijuana reduces the potential for contamination by microorganisms. For added protection, material must be screened for contamination before it is packaged for use as medical marijuana. Since opportunistic infections pose the greatest danger to immunosuppressed med users, marijuana should be sterilized by gamma irradiation. Lastly, consumers should be given careful instructions to ensure their marijuana does not become contaminated prior to use.

Preventative Sprays (Botanical)

Research has shown that, toxigenic fungi are sensitive to the 12 essential oils, and particularly sensitive to thyme and cinnamon. The oils of thyme and cinnamon at ⩽500 ppm have been shown to completely inhibit fungi. 1

Additional research has shown that the growth of a toxigenic strain of Aspergillus flavus decreased progressively with increasing concentration of essential oils from leaves of Cinnamomum camphora and rhizome of Alpinia galanga incorporated into SMKY liquid medium. The oils significantly arrested aflatoxin B1 elaboration by A. flavus. The oil of C. camphora completely checked aflatoxin B1 elaboration at 750 ppm (mg/L) while that of A. galanga showed complete inhibition at 500 ppm only. The oil combination of C. camphora and A. galanga showed more efficacy than the individual oils showing complete inhibition of A. flavusproduction even at 250 ppm. 2

1. K.M Soliman. R.I Badeaa (2002) Effect of oil extracted from some medicinal plants on different mycotoxigenic fungi

2. Bhawana Srivastava, Priyanka Singh, Ravindra Shukla and Nawal Kishore Dubey (2008) A novel combination of the essential oils of Cinnamomum camphora and Alpinia galanga in checking aflatoxin B1 production by a toxigenic strain of Aspergillus flavus

(More to be added soon – bookmark this page)

Pesticides/Insecticides

This is a huge area of concern – you will find over 20,000 words on this subject here


Adaptive Hypotheses

I. Intake-efficiency hypothesis

The intake-efficiency hypothesis is based on the predictions of simple optimal foraging models, which have proven robust for herbivores (Sih and Christensen 2001 ). This hypothesis states that selection favors herbivory over animal-containing diets because herbivorous organisms maximize energy intake by minimizing the energy and time spent searching for and subduing prey. Further, aquatic herbivores may use their food source as habitat (Brönmark and Vermaat 1998 ), or seek refuge in aquatic vegetation associated with their preferred food source (e.g., submerged vegetation and epiphytic algae, respectively Alvarez and Peckarsky 2013 ), thereby decreasing energy expenditures related to locomotion (Cummins 1973 ) and/or predator avoidance. Therefore, the net energy gained from a herbivorous diet may be greater than a diet comprised of metazoan prey.

Herbivores are constantly grazing in order to meet energetic needs (Simpson and Simpson 1990 , Cruz-Rivera and Hay 2000b ), whereas energetic, physiological, and encounter rate constraints prevent animal-consuming taxa from continuously foraging (Arrington et al. 2002 , Karasov and Martinez del Rio 2007 ). As a result of these different foraging behaviors, herbivores continuously have plant material in their gut and omnivores/carnivores process their food in “batches” (discussed in Karasov and Martinez del Rio 2007 ). Batch processing may be followed by periods of hunger therefore, herbivores are probably more continuously satiated relative to omnivores/carnivores. According to optimal foraging theory, satiated animals expend less energy foraging and more energy doing other activities such as mating (Krebs et al. 1983 ). Therefore, herbivores may gain an adaptive advantage by shifting their energetic focus from foraging to reproducing.

II. Suboptimal habitat hypothesis

The suboptimal habitat hypothesis states that herbivory may be adaptive by allowing organisms to invade suboptimal habitats. Here, the term “suboptimal habitat” is relative to habitats that support high abundance and diversity of secondary consumers. Food web interactions often occur over spatially heterogeneous landscapes (Oksanen et al. 1995 ), or “patches” of varying resource quality and quantity. Therefore, an optimal habitat might be a suboptimal habitat at another point in space or time. In freshwater systems, it is generally thought that habitat patches are strongly influenced by abiotic factors such as nutrient availability and/or disturbance frequency (Pringle et al. 1988 ). Higher trophic levels dominate communities when habitat productivity is increased (e.g., Marks et al. 2000 , Deegan et al. 2002 , Beveridge et al. 2010 ) or when disturbance occurs at low to intermediate frequencies (Marks et al. 2000 ). However, consuming a plant-dominated diet is favored in habitats where animal prey are scarce and plant abundance is high (Chubaty et al. 2014 ), such as those with frequent disturbance. Furthermore, the palatability of plants is thought to play a key role in structuring herbivore populations (Elger et al. 2004 ). The most palatable benthic and phytoplankton species are associated with early stages of succession, because fast-growing plants invest less energy in structural and toxic elements (e.g., Porter 1977 , Elger et al. 2004 ). Elger et al. ( 2002 ) investigated the effects of disturbance and nutrient availability on freshwater plant palatability for herbivorous snails (Lymnaea stagnalis) and found that increased disturbance frequency, but not nutrient availability, positively influenced food availability for herbivores (Elger et al. 2002 ), providing evidence for an herbivore advantage in disturbed habitats (e.g., suboptimal habitats).

Classic optimal foraging theory (i.e., optimal diet) predicts that if a resource is abundant, specializing on that resource is preferred (see Chubaty et al. 2014 ). These predictions are supported by early food preference studies, which suggest that herbivores evolved in response to food availability rather than food value (Paine and Vadas 1969 ). Using an evolutionary simulation model, Chubaty et al. ( 2014 ) examined how quality and availability of plant and animal prey shapes the evolution of diet. Results indicate that relative availability of resources can predict an individual's trophic level (Chubaty et al. 2014 ). More specifically, an increased abundance of plants increases herbivore abundance relative to carnivorous animals (Chubaty et al. 2014 ) demonstrating that herbivory may be adaptive when plants are abundant and prey are not (e.g., suboptimal habitats).

Seasonality can also influence habitat quality and resource availability. Organisms are limited to resources that are immediately available. Constant and seasonally varying food supplies are known to influence life histories of many aquatic consumers by altering individual growth and reproduction (output, patterns, mode, etc.). The effects of seasonal food limitation have been well studied in Daphnia (Tessier 1986 , Chapman and Burns 1994 ) and other cladocerans (DeMott and Kerfoot 1982 , Boersma and Vijverberg 1996 ). More specifically, constant food supplies are known to increase growth and brood size of cladocerans. However, food supplies vary in nature and herbivores may gain an advantage by consuming different species or by switching between green, detrital, and/or animal diets seasonally, thereby reducing the effects of specializing on a single food type (Kitting 1980 , Sanders et al. 1996 , DeMott 1998 , Cruz-Rivera and Hay 2000a ).

Herbivory may allow organisms to minimize interspecific competition (via decreased niche overlap) by invading and establishing populations in suboptimal habitats. For example, the globally invasive golden apple snail (Pomacea canaliculata) specializes on freshwater macrophytes and has established successful populations in areas that are uncolonized by other phylogenetically similar species. Further, invading a suboptimal habitat may allow herbivores to escape predation. Trade-offs between foraging and predator avoidance in aquatic consumers are well documented (reviewed by Milinski 1985 ). Camacho and Thacker ( 2013 ) showed that freshwater amphipods exposed to fish predators sought refuge in toxic cyanobacterial mats. Further, amphipods exposed to predators showed higher survivorship on toxic mats as compared to non-toxic mats. These results suggest that herbivores at risk from predators benefit by seeking refuge in suboptimal habitats. If herbivores benefit from invading suboptimal habitats by avoiding predation, equally performing herbivores could be aggregated in both high- and low-quality patches as predicted by an “ideal free distribution” (Fretwell and Lucas 1970 ). Therefore, the ability to colonize and persist equally in both inferior and relatively superior habitats can promote survival of herbivores by exploiting niche opportunities that are unavailable to carnivorous species.

III. Heterotroph facilitation hypothesis

The heterotroph facilitation hypothesis states that herbivory is adaptive because herbivores indirectly consume heterotrophic microbes (bacteria, fungi, and/or protozoa) that are associated with primary producer communities. It has been shown that aquatic herbivores supplement their diets with essential nutrients originating from heterotrophic bacteria (Bowen 1984 , Smoot and Findlay 2010 , Belicka et al. 2012 ) and a strong positive correlation between primary production and bacteria has been documented in several aquatic systems (Cole 1982 ). In limnetic waters, heterotrophic microbes largely contribute to planktonic biomass and are under strong grazing pressure by zooplankton (Arndt 1993 ). Benthic algae in close association with heterotrophic microbes come in several forms (collectively called “periphyton”) and are the primary food source for herbivores in benthic systems (Wetzel 2001 ).

Relative to algae, heterotrophic bacteria are superior competitors for phosphorus (P), incorporating the nutrient into their cell walls (Martin-Creuzburg et al. 2011 ) therefore, these microbes are a rich source of the limiting nutrient for herbivores (Martin-Creuzburg et al. 2011 ). Although P is important for metazoan growth (Sterner and Elser 2002 ), diets composed only of heterotrophs are of poor quality for Daphnia magna suggesting that herbivores may rely on other dietary items for essential biochemicals such as sterols (e.g., invertebrates) or fatty acids (Martin-Creuzburg et al. 2011 ). For example, growth rates of Daphnia magna increased when fed heterotrophic bacteria supplemented with sterols (important for molting) relative to growth of those fed only bacteria (Martin-Creuzburg et al. 2011 ). Related studies found that Daphnia require a diet composed of at least 50% green algae to compensate for a sterol deficiency (Martin-Creuzburg et al. 2005 ). In a vertebrate example, the sailfin molly (Poecilia latipinna) was shown to assimilate both algal material and fatty acids derived from heterotrophic bacteria (Belicka et al. 2012 ). Consumption of heterotrophs along with consumption of autotrophs may allow herbivores to obtain adequate amounts of both P and fatty acids for growth and other life processes, respectively.

IV. Lipid allocation hypothesis

The lipid allocation hypothesis states that herbivory is adaptive because higher consumption of algae with high lipid concentrations may increase fitness. Algae are primary producers of essential lipids that cannot be synthesized by metazoans, but are necessary for their survival (Ahlgren et al. 1990 , Sargent et al. 1995 , Sharathchandra and Rajashekhar 2011 , Guo et al. 2016 ). Although animal prey are rich in lipids relative to algae, wild-caught herbivorous fishes have higher lipase activities in the gut than carnivores, suggesting that lipids are of major importance to herbivores (Nayak et al. 2003 , Drewe et al. 2004 , German et al. 2004 ).

Fatty acids can be incorporated into lipid bilayers of metazoan cells (phospholipids Karasov and Martinez del Rio 2007 ), can serve as precursors for important animal hormones (Brett and Muller-Navarra 1997 ), and can be stored as energy (Wiegand 1996 ) in aquatic consumers. Excess carbon that does not originate from fatty acids can also be stored as lipid reserves in primary consumers (e.g., Daphnia: Sterner and Hessen 1994 , Gulati and DeMott 1997 ), emphasizing the importance of lipid storage. In aquatic organisms, a primary role of lipids is energy storage for reproductive purposes, as they are the main components of ova (Brooks et al. 1997 ). During reproductive periods, lipid compounds are mobilized to the gonads in fish (Wiegand 1996 , Guler et al. 2007 , Wang et al. 2013 ) and increased dietary lipids (from 12% to 18%) result in increased fecundity (Durray et al. 1994 ). Lipid ingestion from algal sources has also been shown to positively correlate with reproductive success in several aquatic organisms (Daphnia, copepods, fishes) and with clutch size in particular (Goulden et al. 1982 , Tessier et al. 1983 , Schmidt and Jonasdottir 1997 , Weers and Gulati 1997 , Martin-Creuzburg et al. 2008 , Guo and Xie 2011 ). In addition, organisms consuming diets rich in phospholipids allocate dietary P to ova (e.g., copepods, Laspoumaderes et al. 2010 ), thereby contributing to offspring growth and survival. Dietary phospholipids are the main constituents of embryonic yolk (Wiegand 1996 ) and thus serve as both an energy source and a component of structural growth in developing embryos (Bell 1989 , Wiegand 1996 ). Furthermore, phospholipids are abundant in the membranes of neural tissues and are thus integral for growth of larvae, which have a high percentage of neural tissue relative to their body mass (Bell et al. 1997 ). As lipids (and phospholipids) are important for storage, structure, and reproduction of aquatic organisms, herbivory may be favored over ominivory and carnivory if essential lipids are obtained from available algal sources.

V. Disease avoidance hypothesis

The disease avoidance hypothesis maintains that herbivory is advantageous because it reduces disease transmission via animals. Many secondary consumers such as piscivores are definitive hosts for parasites, with primary consumers (i.e., invertebrates or small vertebrates) serving as intermediate hosts (Covich et al. 1999 , Marcogliese 2002 ). Furthermore, phylogenetic relatedness and similarity in biological traits between hosts has been shown to be a useful predictor of parasite prevalence in many taxa (see discussion in Huang et al. 2014 ). Specifically, carnivores that are phylogenetically and ecologically similar were shown to harbor similar parasite assemblages (Huang et al. 2014 ), suggesting that diet affects the probability of parasitic infection. Furthermore, a meta-analysis by Choudhury and Dick ( 2000 ) showed that freshwater piscivorous fishes have rich parasite communities as compared to herbivores and zooplanktivores (Choudhury and Dick 2000 , see Dogiel et al. 1961 , for examples). Although herbivores can contract a variety of parasites that do not originate from the diet (see Hoffman 1999 for a full review) and can experience negative effects as an intermediate host (Plaistow et al. 2001 ), herbivory may mediate the effects of animal-facilitated parasites and thus energy allocation to maintenance mechanisms that respond to such parasites.

Alternatively, consuming animal prey may facilitate the transmission of prions, also referred to as transmissible spongiform encephalopathies. These infectious agents are composed of protein and are responsible for mad cow disease in mammals (Dalla Valle et al. 2008 ). Although prions are not as common in aquatic systems as they are in terrestrial systems, prions have been discovered in some fish species (Rivera-Milla et al. 2003 , Dalla Valle et al. 2008 ). Animal tissues are built from proteins that are potentially harmed by these agents, thereby posing a significant threat to aquatic food webs. Because basal items are not protein-rich resources (Mattson 1980 , Sterner and Elser 2002 ), herbivores may benefit from reduced exposure to infectious prions that could alter the functioning proteins comprising their somatic tissues.


SUNLIGHT HELPS US LIVE LONGER

Dr. Jack Kruse and Dr. Alexander Wunsch are the two leading experts and public sources of information on light and its impact on human health. Both have such detailed and scientific explanations of how light works that it takes a while to weed through the details and understand their points.

The bottom line is that spending time in the right LIGHT ENVIRONMENT massively impacts your health and longevity.

In the post below that Dr. Kruse made on LinkedIn, he claims that cell division, cell time, and cell life is based on signals from light and thus impacts our longevity.

From Jack Kruse’s LinkedIn post in 2017:

“In 1964, John Ott along with Dr. Irving Leopold studied the effects of light on rabbit retinal pigment epithelial cells. They documented that that retinal pigment epithelial would not divide unless they were exposed to low levels of ultraviolet light. Roeland van Wijk research has shown that every cell on Earth has to release ELF-UV light from its surface to stimulate mitosis. What this implies is that UV light is necessary for our health because the RPE in the human retina controls our eye clock! The eye clock is what controls our circadian rhythms in our cells and they all link coherently using light water and magnetism to control growth and metabolism. The effect is massive on our lifespan.”

There is so much to light that most people do not understand. Sunlight can heal and indoor light can be toxic. But you have to dig deeper to fully understand the nuances. Ultimately, you are best by spemding most of your time in a naturally lit environment, with as little between you and the outdoors as possible.

At night, this is even more critical. Bathing yourself in artificial light at night is carcinogenic. That's why shiftwork is actually a classified carcinogen. (r)


Promotion of plant health

Disease suppression

Bonanomi et al. (2018a) recently reviewed the functionality of organic amendments to suppress diseases. They concluded that there is a “lack of knowledge regarding the chemical, biochemical, and biological factors responsible for effective organic amendment-based disease suppression”, a main reason being an inadequate “understanding of the feeding preference, e.g., during the saprophytic phase of either pathogenic or beneficial microbes”. Nevertheless, some organic amendments are known to be effective for disease suppression. For instance glucosinolates from green manures of the Brassicaceae, are precursors of toxic isothiocyanates. Their mode of action is supposed to be direct, nematicidal (Vervoort et al. 2014). Most other OM effects on disease suppression, however, are indirect: through, for instance, effects on the activity of saprotrophic, non-pathogenic soil biota.

A meta-analysis to characterize suppressive amendments found very little parameters consistently related to disease suppression (Bonanomi et al. 2010). The C:N ratio of amendments was poorly correlated with suppressiveness, and their decomposition could increase or decrease suppressiveness. Similarly, in an attempt to relate characteristics of dissolved OM (the bioavailable source of C for soil microorganisms) to soil general disease suppression, no consistent relation was found (Straathof 2015). Suppression of the root rot-causing fungus Rhizoctonia solani by volatiles produced by the soil microbial community in Dutch soils was positively correlated with SOM content, microbial biomass and proportion of litter saprotrophs in the microbial community (Van Agtmaal et al. 2018) which partly corroborates the finding of Bonanomi et al. (2010) that general microbial parameters (such as respiration, microbial biomass) are more informative predictors of disease suppression rather than chemical ones.

Plant-growth promotion

Soil OM has been reported to be beneficial for plant, and especially root growth. Beneficial effects on shoot performance have also been reported and some commercial products, such as humic substances, are intended for foliar application (Olaetxea et al. 2018). Lyons and Genc (2016), however, reported that most of the evidence for beneficial effects is anecdotal rather than rigorously mechanistic. Elucidating relations between properties of those substances and their eco-functionality has been difficult, partly for analytical reasons as classical extraction of humic substances may provide a biased view of properties of the various fractions that could then be individually assessed. There is also uncertainty whether the properties of those extracts are due to the chemical structure of these humic substances or whether microbial hormones are entrapped in these supramolecular associations. It was also shown that the pH of the humic extract exerted different hormonal effects: acid extracts exhibited auxin-like activity (i.e. effects on the size and architecture of the root system), whereas at neutral pH the extract exhibited gibberellin-like activity (i.e. conversion of starch into sugars, and with auxins stimulation of stem elongation). Most attention to date has been given to auxins, especially indole acetic acid (Nardi et al. 2018). From a management perspective it has become clear that the properties of these organic substances are not or hardly related to their provenance (Garciá et al. 2016). Plants that benefit from these substances may also be more tolerant or resistant to soil-borne pathogens.


RESULTS

Tree biomass and soil C stocks are inversely related across the forest line

The multi-stemmed Betula pubescens ssp. czerepanovii (arctic downy birch) trees had a density of about 12 trees per 100 m 2 in the forest, corresponding to 1.2 kg C m −2 (Fig. 1). In the forest, B. pubescens was the only ectomycorrhizal species, but several ectomycorrhizal plants contributed to the vegetation at the forest edge and above the tree line. The highest coverage of ectomycorrhizal species in the field vegetation, that is, excluding trees, across all sites, was found in the shrub tundra, where Betula nana (dwarf birch) was abundant (Fig. 1a Table S5). However, the field vegetation was dominated by ericoid mycorrhizal dwarf shrubs at all sites, and their total coverage was higher in the forest than in the heath. The total C pool in the vegetation doubled from just under 1 kg C m −2 in the heath to around 2 kg C m −2 in the forest (Fig. 1b). The total C pool in the organic soil layer, however, decreased from more than 6 kg m −2 in the heath to about 2 kg m −2 in the forest. The litter layer amounted to 283 ± 17 g C m −2 across all sites, and the humus layer accounted for 88% (forest) and 95% (other sites) of the organic soil C stocks (Fig. 1b).

The average C-to-N ratio of the entire organic horizon was higher in the forest (27.5 ± 0.8) than in the other sites (18.9–20.1 F = 14.0 P < 0.001 Fig. S2) and correlated negatively with C stocks (Fig. S3). Higher birch tree densities were related both to smaller soil C stocks and higher C-to-N ratios in the organic layer across all plots (r 2 = 0.74 and r 2 = 0.66, respectively P < 0.01) and among forest plots only (r 2 = 0.54 and r 2 = 0.61, respectively P < 0.01 Fig. S2). However, coverage of ectomycorrhizal plants in the field vegetation correlated positively with soil C stocks across all plots (P < 0.01 Fig. S3) or showed no relationship with C stocks when excluding the forest site (r 2 = 0.11, P > 0.05).

Carbon–nitrogen dynamics

Fresh litter inputs (uppermost L1 layer) had highest C-to-N ratios in the heath and shrub, and C-to-N ratios decreased with depth from the fresh litter (L1) to the fragmented litter layer (L2, second layer) and the upper humus layer (H1, third layer) at all sites (Fig. 2a Table S6). The C-to-N ratios kept decreasing with humus depth (H1–H3) in the heath, shrub and forest edge. In the forest, in contrast, C-to-N ratio reached a minimum in the uppermost humus (H1) and then rose again in deeper humus layers (H2–H3). While δ 13 C patterns were similar for all sites (Fig. 2c), δ 15 N showed a steeper increase with depth in the forest and forest edge than in the shrub and heath (Fig. 2b).

Fungal biomass (as inferred from ergosterol concentration) generally declined with depth. Fungal biomass in the upper three layers (L1, L2 and H1) was about half in the heath compared to the other sites (Fig. 2d Table S6). Mycelial growth was about 10 times higher in the forest than in the heath (Fig. 2i), while mycelial C-to-N ratios (ranging from 17.3 to 19.2), δ 13 C (−26.3 to −27.5) and δ 15 N (0.2–2.2) were similar across sites (n = 1–6) and matched levels previously measured in ectomycorrhizal fungi (Clemmensen et al. 2006 ).

Dissolved organic C and N concentrations were highest in forest, and the ratio between these pools was lowest in the heath (Fig. 2e,f,g). Inorganic N concentrations were highest in the heath (Fig. 2h), and heath and shrub had higher abundances of bacteria and archaea involved in inorganic N transformations (Fig. 2k,l). Total bacterial-to-fungal ratios also seemed higher in the heath as indicated by the 16S-to-ITS copy ratio (Fig. 2j).

Fungal communities

Fungal community composition varied along three independent gradients. At all sites, communities shifted with soil depth, with saprotrophic fungi dominating in litter layers and mycorrhizal and other root-associated fungi dominating in humus layers (along the second CA axis explaining 8.3% of variation Fig. 3a,b). Fungal communities also shifted along the vegetation gradient from heath to forest, both in the humus (along the first CA axis 9.8% Fig. 3a,b) and litter layers (along the third CA axis 4.8% Fig. S4). CCA analysis confirmed the significance of differences in communities among sites and depths (Table S7).

The relative abundance of root-associated Ascomycota (including ericoid mycorrhizal fungi) was highest in the forest, whereas the relative abundance of ectomycorrhizal fungi was highest in the heath and shrub habitats (Fig. 3b,c Table S6). The share of ectomycorrhizal fungi with less differentiated mycelia (short-distance exploration types) was higher in the heath and shrub heath, while the share of ectomycorrhizal species with mycelia differentiated for long-distance transport increased towards the forest (Fig. 3c Table S6). Long-distance ectomycorrhizal species belonging to the genus Cortinarius were particularly abundant in the forest and forest edge humus, but Leccinum and Piloderma species were also common, while short-distance ectomycorrhizal species, particularly Inocybe and Tomentella species, dominated communities in the heath and shrub humus (Fig. 4). Communities of other root-associated fungi in humus also shifted gradually along the vegetation gradient, but whereas the relative abundance of root-associated Basidiomycota (Sebacinales) was higher in heath and shrub, there were no clear shifts among Ascomycota classes (Fig. S5). The most well-characterized ericoid mycorrhizal fungus Pezoloma ericae (earlier named Hymenoscyphus ericae) was most abundant in forest.

Within the saprotrophic communities in the litter layers, moulds increased towards the heath and shrub, whereas some Basidiomycota genera were only found among the dominant fungi in the forested sites (Sistotrema, Trechispora and Luellia), while Mycena species were found along the entire vegetation gradient (Fig. S5).

Tree root exclusion decreases decomposition

After 3 years of field incubation, both forest and heath litter substrates were dominated by free-living saprotrophic fungi, whereas humus substrates were dominated by root-associated fungal communities, although forest humus also had a large proportion of moulds and yeasts (Fig. 5a). Mass loss, respiration and fungal biomass were overall higher in litter than in humus. First year mass loss was faster for forest litter than for heath litter but, slower for forest humus than for heath humus (Fig. 5b,c,d Tables S8 and S9). Exclusion of living birch roots decreased fungal biomass slightly and almost eliminated ectomycorrhizal fungal colonization of the decomposition bags, while other root-associated fungi, including ericoid mycorrhizal ones, remained unaffected (Fig. 5a,c Table S8). Presence of living roots overall increased mass loss of both litter and humus substrates after 3 years (Fig. 5b Table S9). While litter substrates continued to decrease in mass throughout the incubation, the mass of humus samples increased between years 1 and 3, particularly in plots without living birch roots (Fig. 5b Table S9).


Experimental procedures

Construction of Pht1 promoter-reporter gene fusions

Pht1 promoters were cloned as transcriptional or translational fusions to the green fluorescent protein (GFP) or β-glucuronidase (GUS) reporter genes in the binary vectors pBI101.3 (Clontech Laboratories, Palo Alto, CA, USA) and pBI101.3-gfp, respectively. The latter vector was constructed by excising the GFP coding sequence and nopaline synthase terminator from pGEM.Ubi1-sgfpS65T ( Elliott et al., 1999 ) using SacI and BamHI, blunting the SacI end using Klenow polymerase, and cloning this into pBI101.3 which had been digested with EcoRI, blunted with Klenow polymerase, and digested with BamHI.

DNA from A. thaliana ecotype Columbia was isolated using the Qiagen DNeasy kit (Qiagen, Pty. Ltd. Clifton Hill, Victoria, Australia), and used as a template to amplify the Pht1 promoter fragments. The promoters were amplified using Pfu high-fidelity polymerase (Stratagene, La Jolla, CA, USA) or Expand High Fidelity polymerase (Roche Diagnostics Australia, Castle Hill, NSW, Australia), according to the manufacturer's instructions. Primers were designed to amplify the promoter lengths shown in Table 1, and restriction sites were incorporated in the primers to facilitate cloning into the binary vectors. Further information on cloning strategies is available on request from the authors. Where present, introns in the untranslated leader sequence were included in the promoter-reporter constructs.

Plant transformation and growth conditions

Binary vectors were introduced into Agrobacterium tumefaciens strain AGL1 ( Lazo et al., 1991 ) by electroporation. Transformation of A. thaliana was done using the floral dip procedure ( Clough and Bent, 1998 ), and transgenic seedlings were selected on 0.5 × MS medium ( Murashige and Skoog, 1962 ) containing 50 µg ml −1 kanamycin. At least 10 independent transgenic lines were generated for each construct.

Plants were grown at 24°C with a 16-h photoperiod and light intensity of 150 µmol m −2 sec −1 . To test for promoter induction by phosphate deprivation, T1 or T2 progeny were germinated in a soil/sand mix containing high or low phosphate levels, and grown for 3 weeks. The low phosphate mix contained one part soil with low phosphorus (10 mg total P per kg) and nine parts fine washed sand. To create the high phosphate mix, finely powdered Ca(H2PO4)2.H2O was mixed through the above soil/sand mix at a rate of 60 mg phosphorus per kg of soil/sand mix. Plants growing in this soil/sand mix were watered with a nutrient solution containing 500 µ m Ca(NO3)2, 500 µ m KNO3, 250 µ m MgSO4, 18 µ m NaFeEDTA, 45 µ m H3BO3, 4.5 µ m MnCl2, 315 n m CuCl2, 750 n m ZnCl2 and 15 n m (NH4)6Mo7O24, but no Pi.

Pollen germination was carried out as described by Li et al. (1999 ).

Reporter gene assays

For reporter gene assays on soil-grown plants, the plants were carefully removed from the pots, soil was washed off the roots with water, and GFP fluorescence was observed in intact seedlings using a Leica MZ6 dissecting microscope with the GFP PLUS fluorescence module (Leica AG, Heerbrugg, Switzerland). Images were collected using a SPOT digital camera and associated software (Diagnostic Instruments, Sterling Heights, MI, USA). Confocal microscopy on intact roots was done using a Leica TCS SP2 confocal on an upright Leica DMRXE microscope.

Histochemical detection of GUS activity was done using the substrate 5-bromo-4-chloro-3-indolyl glucuronide (X-gluc Jefferson, 1987 ), by vacuum-infiltrating seedlings in assay buffer (50 m m sodium phosphate pH 7.0, 0.1%Triton X-100, 0.5 m m potassium ferrocyanide, 0.5 m m potassium ferricyanide, and 10 m m EDTA) containing 0.05% X-gluc, and incubating at 37°C for 30 min to overnight. Green tissues were destained with ethanol prior to observation. For sectioning, tissue was briefly fixed in FAA (10% formalin/5% acetic acid/50% ethanol), dehydrated in ethanol, briefly equilibrated in Histoclear (National Diagnostics. Atlanta, GA, USA) and embedded in Paraplast embedding medium (Sigma-Aldrich Corp., St Louis, MO, USA). Embedded tissue was cut into 10 µm sections using a rotary microtome.

Quantitative fluorimetric GUS assays were done using the substrate 4-methyl umbelliferyl glucuronide (MUG) as previously described ( Jefferson, 1987 ). Fluorescence was measured using a Fluoroskan Ascent microtitre plate reader. Protein concentrations were measured using the Bradford assay kit (Bio-Rad Laboratories, Hercules, CA, USA).

RT-PCR analysis

RNA was isolated from a range of A. thaliana tissues, including cotyledons and young leaves from 3-week-old soil-grown plants, old leaves from 5-week-old soil-grown plants, flowers, green siliques, roots from 3-week-old plants grown in high Pi soil, and roots from 3-week-old plants grown in low Pi soil, using an RNeasy kit (Qiagen). Approximately 1 µg of RNA was used as a template for first strand cDNA synthesis, using a Superscript First Strand cDNA Synthesis kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. Contaminating genomic DNA was removed from the RNA samples using DNase 1 (Qiagen).

One µl of first strand cDNA was then used for PCR using gene-specific primers for each member of the Pht1 family except Pht11 and Pht12, which were both amplified with the same primer set. Primers were positioned on either side of introns when introns were present within the coding sequence. Details of primer sequences are available from the authors on request. PCR was carried out using EXPAND High Fidelity polymerase (Roche) according to the manufacturer's instructions, using 2.2 m m MgCl2 in 50 µl reactions. Thermal cycling consisted of an initial denaturation at 94°C for 2 min, followed by 10 cycles of denaturation at 94°C for 15 sec, annealing at 50°C for 30 sec, and extension at 72°C for 2 min, and then an additional 20 cycles during which the extension time was increased by 5 sec per cycle, followed by a final extension at 72°C for 7 min. The expected sizes of products amplified from cDNA and genomic DNA are shown in Table 2.

Gene Expected product from cDNA (bp) Expected product from genomic DNA (bp)
Pht11 + Pht12 357 508
Pht13 358 482
Pht14 391 391
Pht15 679 777
Pht16 579 579
Pht17 453 453
Pht18 418 3483
Pht19 494 1654