Understanding Colony Forming Units (cfu) and Colonization

cfu

It is important to note that beneficial bacteria and/or fungi numbers (expressed through cfu) are critical in determining the efficiency in root pathogen suppression/control.

In simple terms, high densities of viable and rhizosphere competent microbes that can achieve rapid colonization of the substrate are required for efficient pathogen control. Therefore, low cfu of beneficial bacteria and/or fungi will exert limited to no control over pathogens while high cfu of beneficial microorganisms will exert high levels of control over pathogens. Based on this, it is critical in root disease prevention that a high cfu of beneficial microorganisms are applied to the substrate and roots, and that these beneficial microorganisms are able to efficiently colonize the rhizosphere. Colonization comes down to several factors; 1) high numbers of beneficial microorganisms need to be applied to the substrate/root zone to ensure high numbers of the beneficial microorganisms are present in the substrate/root zone; 2) the substrate that the microorganisms are applied to needs to be conducive to the survival and colonization of the beneficial bacteria and/or fungi species that have been applied; this comes down to physical (e.g. air water properties, temperature, media matrix) and chemical (e.g. nutrient status, EC, pH, carbon to nitrogen ratio) properties of the substrate and 4) plant type, inline to microorganism species is important. That is, a symbiosis occurs between the plant’s roots and the beneficial microorganisms. Some beneficial microorganisms will colonize very efficiently in one crop type while colonizing far less efficiently in another crop type.

Colonization

The ability of a beneficial bacteria or fungi species to multiply and colonize is critical in root zone disease prevention because key to the mechanisms of control that beneficial bacteria and fungi exert over pathogens is competition for ecological niches. What this basically means is that if beneficial microorganisms are able to colonize the root zone in high numbers they outcompete pathogenic microorganisms, leaving little to no opportunity for pathogens to gain a foothold.

Why Synergy Coir for Coir Growers?

Synergy coir contains high cfu (guaranteed minimum analysis of 1.5 billion cfu per gram) of carefully selected, rhizosphere competent Trichoderma species (sp.). Trichoderma sp. have been shown in numerous studies to improve resistance of plants against disease by acting as direct competition for ecological niches against pathogens, changing plant cell wall compounds, increasing enzyme activities, and/or production of pathogenesis-related proteins.

Trichoderma species are considered by many to be the superhero (super fungus) against evil parasites.[1] They are highly adept assassins of pathogens.

Trichoderma sp. are shown in numerous studies to colonize extremely efficiently in coir. In fact, coir is shown in several studies to be the ideal substrate for the mass production/multiplication of Trichoderma species. In simple terms, Trichoderma sp. thrive in coir creating a prolific, dynamic, beneficial microflora that acts to prevent root disease.

Trichoderma sp. are emerging as one of the most important ‘Benny’ species in the battle against root pathogens in hydroponic environments. Many Trichoderma species are specific for certain pathogens and will eliminate them if they are present in the same location. This is achieved through a number of different processes. The main method of attack is that the Trichoderma coil around the pathogenic fungi, release enzymes to break down the cells and consume its prey. Trichoderma also release a number of antibiotic compounds for direct control. Another method of pathogen control is through inducing systemic resistance in the host plant. Trichoderma does this by invading the plant’s root system to the depth of a few cells, which triggers the plant to launch its natural defence mechanism to wall off the Trichoderma, in doing so the systemic resistance spreads through the entire plant so that foliar and fruit diseases may be controlled as well.

Trichoderma sp. produce lytic enzymes (cellulose, hemicellulose, amylase, lipase, pectinase, chitinase, protease and xylanase). Recently, the role of extracellular enzymes has been documented by several researchers where pathogen suppression/control is based on the secretion of a rich and multiple mixture of cell wall degrading enzymes able to hydrolyze the cell walls of various plant pathogens. Among others, chitinase, glucanases, and proteases have been described as principal components of the multienzymatic system of Trichoderma strains. Of notable importance is that Trichoderma release chitinase enzymes that break down chitin—the primary material that makes up the cell walls of pathogenic fungi. The chitinase enzymes released by Trichoderma lyse (penetrate) the cell walls of the pathogenic fungi and, in turn, this protects the roots from being attacked. In fact, when a large population of pathogenic fungi exists in the soil or substrate, Trichoderma increase chitinase production and feed almost exclusively on the pathogens.

Trichoderma also release another enzyme that is highly beneficial in root disease prevention: cellulase.

Cellulase is beneficial in two ways. Firstly, cellulase aids in the breakdown of cellulose in soils and substrates, turning it into simple sugars (beta glucose). These simple sugars act as an energy/food source for beneficial bacteria and fungi. Notably, plant root systems consist of, among other compounds, high levels of cellulose while coir consists of about 35% cellulose in its structural makeup. Therefore, the cellulase enzymes produced by trichoderma, act much like the liquid enzyme products purchased through hydroponic stores – in that the cellulase (enzyme) breaks down cellulose (root and coir substrate matter) resulting in the creation of simple sugars which act as an energy source (food) for beneficial bacteria and fungi.

Additionally, cellulase can penetrate root cells. When the cellulase penetrate the root cells, they automatically trigger the plant’s natural defense system. The plant’s metabolism is stimulated, but no real harm is caused to the plant. In this regard, Trichoderma has a synergistic relationship with plants. Trichoderma feed on sugars and carbons secreted by roots (termed *root exudates), while the plants develop a heightened resistance against pests and pathogens.

*Root exudates: Plants release organic acids including amino acids, carbohydrates, secondary metabolites and sugars through the roots; these acids, carbohydrates, secondary metabolites and sugars are referred to as root exudates. These chemical compounds attract microorganisms to the rhizosphere. In turn, these plant-associated microorganisms, via different mechanisms, influence plant health and growth.

Approximately 20 to 40% of photosynthetic carbon fixed by a plant is released as root exudate into the surrounding soil or substrate, attracting microorganisms and creating a dynamic ecological niche known as the rhizosphere. The rhizosphere represents a microbial hot spot, in which positive, neutral and negative interactions take place with the host plant which can affect plant health and growth. Plant growth and productivity is heavily influenced by the interactions between plant-roots and the surrounding soil or substrate, including the microbial populations within the soil/substrate. The plant rhizosphere harbours microorganisms that may have positive, negative or no visible effect on plant growth. Although most rhizospheric microbes appear to be benign, deleterious microorganisms include pathogens and microbes producing toxins that inhibit root growth or those that remove essential substances from the soil. By contrast the main mechanisms for plant growth promotion include suppression of disease (biocontrol); enhancement of nutrient availability (biofertilization); and production of plant hormones.

[1] F.A. Mohiddin, M.R. Khan, S.M. Khan and B.H. Bhat, 2010. Why Trichoderma is Considered the Super Hero (Super Fungus) Against Evil Parasites? Plant Pathology Journal, 9: 92-102.

The Complexities of Beneficial Bacteria and/or Fungi Bioinoculants

The problem with beneficial bacteria and fungi additives (technically termed bioinoculants or microbial inoculants) is that the science is extraordinarily complex, so much so that experts in the field of microbiology are grappling to come to terms with the optimum use of bioinoculants in agriculture. So, for example, one species of bacteria or fungi (‘Benny’) may show promise in one environment, but when this species is introduced into another environment it fails to colonize to a level that offers effective pathogen control. In other cases, one species of bacteria or fungi may show promise in combating one species of pathogen while exerting little or no control over another pathogen. Based on this, scientists have experimented with using multiple species of beneficial bacteria and fungi to achieve broad-spectrum pathogen control; however, a combination of species will not necessarily produce an additive or synergic effect, but rather a competitive process where one species of beneficial bacteria or fungi suppresses another. For example, recently, it was demonstrated that Trichoderma harzianum was able to parasitize the mycelium of an arbuscular mycorrhizal (AM) fungus, thus affecting its viability.[1] Similarly, another study found that Trichoderma harzianum was suppressed by Mycelium of the Arbuscular Mycorrhizal Fungus Glomus intraradices in root free soil. Further, the effects of fungi belonging to the genus Trichoderma on spore germination and hyphal growth of Glomus mosseae have been examined in vitro, and contradictory results have been obtained. However, the results from pot experiments suggest that Trichoderma species suppress AM root colonization, although this depends on the timing of inoculation and the host plant species. On the other hand, adverse effects of AM fungi on the population density of Trichoderma koningii have also been observed.[2] In simple terms, the application of Trichoderma sp., in combination with AM fungi (AMF), potentially can limit at least some species of AMF from colonizing and vice versa.

Other issues also present where the success of beneficial bacteria and/or fungi are concerned. For example, one species may thrive in low nutrient environments, while in another environment where high levels of nutrients are present the same species may fail to colonize efficiently. Take, for example, arbuscular mycorrhizal fungi (AMF). A primary effect of AMF symbiosis is improved phosphate (P) nutrition made possible by an extensive hyphal network of AMF. This not only allows the plant to overcome the phosphorous depletion zone around the root surface interface but also allows it to reach immobile P that the AMF can solubilise. This phenomenon is most apparent in low P soils and substrates. However, with increasing bioavailbale P, the benefits of AMF decline and colonization is reduced. In general, the benefits of AMF are lost to plants that have other means of obtaining P from soils or substrates. As a result, low rates of colonization of AMF occurs in high bioavailable P environments.[3]

H.J. Hawkins et al (2004) note that a nutrient medium containing a P concentration of 0.9 mM (27.876384 ppm P) failed to produce viable mycorrhizal colonization.[4] Similar findings by G.Nagahashi (1996) demonstrates that mycorrhizae grown in the presence of P at 1.0mM (30.973 ppm) showed significantly less hypal branching than in lower P environments. [5]

What this really tells us is that while AMF are beneficial additions (as microbial inoculants) to soils and substrates where low P is present, AMF microbial inoculants are less than perfect in hydroponic growing situations where high P is present.

This is important information for growers to understand. Ultimately, success in broad spectrum root disease prevention comes down to 1) applying a species or multiple species to the hydroponic system in high numbers; 2) ensuring the beneficial microbes applied to the system are conducive to the environment in which they are placed; 3) where more than one species is applied it is critical that they are noncompetitive and ideally combinations of species work synergistically to combat pathogens, and; 4) pathogen prevention is highly reliant on the ability of the beneficial bacteria and/or fungi to colonize the substrate and the roots. Should colonization be compromised by a physical factor (e.g. substrate type), a chemical factor (e.g. nutrient status) or a biological factor (e.g. a non-compatible/competitive species of bacteria or fungi) growers leave their plants wide open to root pathogens.

[1] De Jaeger, N. et al (2011) Trichoderma harzianum might impact phosphorus transport by arbuscular mycorrhizal fungi. FEMS Microbiology Ecology, Volume 77, Issue 3, September 2011, Pages 558–567

[2] Helge Green et al. (1999) Suppression of the Biocontrol Agent Trichoderma harzianum by Mycelium of the Arbuscular Mycorrhizal Fungus Glomus intraradices in Root-Free Soil

[3] K.K. Treseder, M.F. Allen. Direct nitrogen and phosphorus limitation of arbuscular mycorrhizal fungi: A model and field test. New Phytol, 155 (3) (2002), pp. 507-515

[4] 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.

[5] G.Nagahashi, D.D.Douds Jr. and G.D. Abney (1996) Phosphorus amendment inhibits hyphal branching of VAM fungus Gigaspora margarita directly through its effect on root exudation

Genetic Potential and the Law of the Limiting Factor

The term “genetic potential” in medicinal crops refers to the amount of production, with regards to quantity (yield potential) and quality (essential oil yield) that a plant has been genetically programmed to produce, if all conditions are exactly perfect.

Therefore, the genetic potential of a plant is expressed under optimum growth conditions where the environment (e.g. temperature, Relative Humidity, CO₂, light, root zone health) and nutrients are maintained within ideal parameters. Failing to provide these optimal parameters will result in a plant producing less than its genetic potential.

This is where the law of the limiting factor comes into play. In short, limiting factors reduce the ability of a plant to achieve genetic potential.

The law of the limiting factor is based on ‘Liebig’s Law of the Minimum’, which states that growth rates are determined not by the total amount of resources available, but by the scarcest resource.  It may be a physical factor such as temperature or light, a chemical factor such as a particular nutrient, or a biological factor such as pests or pathogens. The limiting factor may differ at different times and places. However, if every factor that influences plant growth is within optimal parameters bar one, this single factor will limit growth. Therefore, achieving optimum yields must be seen as a holistic process which is determined by each and every factor (e.g. air temperature, relative humidity, CO2 levels, light quality and quantity, adequate oxygen and moisture in the root zone, root health, plant nutrition).

This is an extremely important principle for growers to understand. Failing to maintain just one single factor at optimum will result in less than optimum yields and quality. Thus, attention must be paid to carefully monitoring and controlling ALL key environmental, biological and plant nutritional factors at optimal levels. Neglecting just one of these factors will result in a plant producing at less than optimum.

Root Disease

Healthy plants equate to optimum yields. Conversely, unhealthy plants produce less than optimum yields. Growing healthy plants that achieve genetic potential comes down maintaining all factors that influence growth within ideal parameters. This means ensuring close attention is paid to factors such as air temperature, Relative Humidity, CO₂ levels, light quality and quantity and plant nutrition (influenced by factors such as pH, EC and nutrient status). Additionally, it also means paying very close attention to ensure ideals are maintained within the root zone of the plant. This one comes down to ensuring, among others, an ideal air water relationship in the substrate is maintained while also ensuring that root zone pathogens are excluded from the growing system.

It has been estimated that between 65 – 80% of all plant health problems start in the roots of plants. Hidden from the human eye, the effects of root disease often go unnoticed until the end of the crop cycle, when damaged roots can result in significant yield losses. 

The problem is that for many growers out of site is out of mind and because the roots of plants are typically hidden from site, root disease prevention isn’t given the attention it deserves.

This means that many growers are harvesting less than optimum yields.  

Root Disease in Med Crops

A recent three year long study in Canada, which looked at Fusarium and Pythium species in the roots of hydroponically grown cannabis found that root rot in tested cannabis crops was caused by two Pythium species – Pythium dissotocum and P. myriotylum, as well as two Fusarium species – Fusarium oxysporum and F. solani.

Following the positive identification of the pathogens, it was important to confirm that these fungal species were in fact responsible for the observed root disease. Pathogenicity tests were carried out using groups of healthy cannabis roots, where one group served as a control and the other groups were inoculated with one of the fungal species.

The roots that were inoculated with P. dissotocum or P. myriotylum experienced visibly stunted growth, as well as a browning and rotting of the roots, in concordance with the originally observed diseased plants. P. aphanidermatum was observed to cause damping-off of the cannabis plants. Roots inoculated with F. oxysporum and F. solani exhibited browning and root rot, with F. solani appearing to be the more aggressive of the two species.

Analysis revealed that the Fusarium species affecting cannabis plants shared 99–100% genetic ancestry with isolates causing stem rot and wilt in other hosts, including cumin and tomato, suggesting they were not uniquely adapted to cannabis. The potential for spread of F. oxysporum through the hydroponic system was confirmed by its detection in the recirculating nutrient solution. Furthermore, rooted cuttings obtained from commercial propagators were found to harbour Fusarium root infection that resulted in subsequent stunting, yellowing and occasional death of plants. This demonstrates the potential for long-distance spread of the pathogen. The two Pythium species recovered from cannabis plants have an extremely broad host range and are not unique to cannabis. An additional species, P. was recovered from diseased plants grown under greenhouse conditions in 2018.

The main source of initial pathogen contact and spread in hydroponic systems comes from the use of a contaminated hydroponic mineral solution. Unsterilized or poorly sterilized tools, equipment, and tubing can also harbor the harmful pathogens and lead to system-wide contamination.

Read original study

Prevention, not Cure, is Critical where Root Disease is concerned

The most effective way to ensure yield losses don’t occur, as a result of root disease, comes down to prevention rather than cure.

Four key strategies for prevention of root disease should be employed: (1) most critically, root zone oxygen levels should be maintained at levels that ensure roots don’t become oxygen starved; (2) grow room hygiene best practice is critical for preventing the introduction of pathogens into the growing environment (3) growers should always strive to maintain key environmental factors (air temp, water/nutrient/substrate temperatures, light levels and colour spectrum, CO2, RH, nutrition) within ideal parameters to ensure their plants remain healthy (a healthy plant is less prone and more resistant to disease) and; (4) pathogen preventatives/additives such as beneficial microorganisms (bennies) should always be used as a standard (must do) growing practice.

Root Zone Oxygen Levels

In the vast majority of cases where root disease (e.g. root browning, root death) is detected in crops, the underlying cause of the problem is root zone oxygen starvation. I.e. roots growing in poorly aerated media are weaker and more susceptible to micronutrient deficiencies and root rot pathogens than roots growing in well-aerated media.[1] Additionally, high nutrient and substrate temperatures have been shown to cause direct damage to roots even in the absence of plant pathogens.[2] A decrease below 3 or 4 mg L-1 of dissolved oxygen inhibits root growth and roots begin browning which can be considered as the first symptom of the oxygen starvation.[3]

For this reason, the most important step growers can take towards preventing root disease is to ensure that root zone oxygen is maintained at levels where root health is not compromised.

[1] Ingram, D. L., Henley, R. W. and Yeager, T. H. 2003. Growth Media for Container Grown Ornamental Plants.<http://edis.ifas.ufl.edu/pdffiles/CN/CN00400.pdf>.

[2] Alhassaen, K. (2006). Pythium and Phytophthora associated with root disease of hydroponic lettuce. PhD thesis, University of Technology, Sydney, 328pp.

[3] Gislerød, H. R. & Adams, P. (1983). Diurnal Variations in the Oxygen Content and Requirement of Recirculating Nutrient Solutions and in the Uptake of Water and Potassium by Cucumber and Tomato Plants. Scientia Horticultura, Vol.21, No.4, (Dec 1983), pp. 311–321, ISSN 0304-4238

Good vs. Bad Microflora and Root Zone Oxygen

Beneficial bacteria and fungi thrive in an oxygen rich (aerobic) environment – harmful bacteria and fungi do not. By increasing the oxygen levels in the root zone, we promote good bacteria and fungi while creating a hostile environment for harmful microflora.

In simple terms, where oxygen levels are high in nutrients and substrates we promote beneficial bacteria and fungi.

Where oxygen levels are low (anaerobic) in the nutrients and substrates we promote bad bacteria and fungi.

Grow Room Hygiene

Hygienic grow room practices are pivotal in preventing the introduction of pathogens into the growing system. Studies have consistently shown that, second to root zone oxygen levels, unhygienic practices are the primary cause of contamination in hydroponic systems. 

Pathogens are soil inhabitants. This is why hydroponics and soil don’t mix. Avoid introducing soil into your hydroponics environment. This means taking precautions such as not dragging soil from outdoors into your (indoor) growing environment on your shoes, clothes or hands.

Pathogens are found in untreated water. Zoospore-producing microorganisms such as Pythium and Phytophthora are particularly well adapted to aquatic environments. Because of this, untreated water such as stream, dam, and shallow bore water are high-risk when used in hydroponic systems. If you are going to use stream, dam or bore water you will need to sterilise it prior to use .Rainwater should also be treated because of the likelihood of it collecting windblown soil which can carry pathogens.

Dead and decaying plant material should never be left lying around in the grow room. Some pathogens have evolved a strategy of becoming dormant in the dead leaves, stems, and roots where they previously caused disease. Inside these tissues they are protected from the hostile environments of the soil and air and are away from competition with other organisms in the soil and air. They have at hand a ready supply of nutrients when conditions become favourable again. Pathogens such as Pythium can survive for months in plant debris. A disease may recur if infested debris is left in the grow room where it may come in contact with the next crop.

Unsterilized equipment used in the grow room can introduce pathogens into the growing system. For this reason, it worth soaking equipment that comes into contact with the nutrient solution and substrate from time to time in a bleach solution. Irrigation lines, pots etc and the nutrient tank/reservoir should be thoroughly washed and flushed with a strong bleach solution between crop cycles.

Optimum Environmental Conditions for Optimum Plant Health

Pathogens can take hold of a weak, stressed crop far more easily than they can a healthy crop. Therefore, ensuring that your plants remain healthy through the correct nutrition (particularly during heavy fruiting) and optimum environmental conditions (air temp, water/nutrient temp, RH, CO₂ levels etc) will give your plants increased resistance against root pathogens. I.e. plants grown in optimal environmental conditions will be more resistant to root disease than plants that are subjected to stress as a result of less than optimal environmental conditions.

Over-Fertilization and Root Disease

Root diseases are more severe on over-fertilized plants. The cause of this damage is two-fold. First, excess nitrogen suppresses the plant’s natural defense response. Second, the accumulation of salts in the growing medium damages root tips, providing an easy means for pathogens to infect the plant.

Insects can also Introduce Root Disease

Fungus gnats (Sciaridae) and shore flies have been shown to infect plants with Pythium and Fusarium. Additionally, many other insects have been shown to act as vectors for plant diseases and viruses. For example, aphids are known to transmit over 150 different kinds of plant viruses. For this reason, it is critical to keep a close eye on things with respects to insects/pests in and around your crop. Prevention methods such as filtering inlet air before it enters the growing environment can aid in keeping insects outside of the grow room while yellow sticky traps can act as an early warning system for identifying the presence of insects in the grow room. Be vigilant and detect and deal with insects early.

Clones Purchased from Third Party Sources can come with Root Pathogens

This is something growers need to be aware of. When purchasing clones from others always treat them with a root pathogen preventative/curative as soon as possible.

Pathogen Preventatives/Additives are a Must

Growers should always use root pathogen preventatives in their growing system. This comes down to using products such as beneficial bacteria and/or fungi to create a biologically diverse growing system. Application at the earliest point possible is imperative to ensure a healthy microflora in the growing system is established from the outset.

Symptoms of Root Disease

Symptoms of root disease are often first noticed as ‘stunting’ (reduced growth), wilting stems and leaves, or yellowing at leaf tips. Upon further inspection, root disease can be recognized by a browning root system that breaks away when pulled. This may also be accompanied by a musty smell as the root system decays. On the stems of cuttings a soft watery rot may develop.

However, it is important to note that root development and plant growth rates can be impaired long before root disease symptoms become apparent. For example, one study notes:

“A surprising aspect of hydroponic crops with Pythium root rot is that the foliage often appears green and healthy even when root rot has become severe. Foliar discoloration normally develops only when the root systems have become almost entirely rotted. Under controlled conditions, the leaf canopy of pepper plants inoculated with (the pathogens) P. aphanidermatum or P. dissotocum often becomes darker green than that of the noninoculated controls (non-infected crops).” [1]

Thus, because root disease can impact on growth while symptoms might not be apparent preventing root disease before it begins is imperative.

Additionally, root pathogens drain energy from the crop even where signs of root disease may not be apparent; i.e. plants have natural defense mechanisms to ward off attacks from pathogens; however, when a plant comes under attack it directs energy away from growth towards defense; the net result of this process is a reduction in growth due to growth–defense tradeoffs.   Growth–defense tradeoffs are thought to occur in plants due to resource restrictions, which demand prioritization towards either growth or defense, depending on external and internal factors. While the deployment of defense mechanisms is imperative for plant survival, defense activation generally comes at the expense of plant growth.

Therefore, healthy plants, in a least some instances, are able to ward off pathogens; however, energy is directed away from growth, towards defense. As a result, yields suffer. The thing is, this can too easily go unnoticed and as a result some growers are left wondering why their yields are lower than they could/should be.

[1] Sutton, J. et al (2006) Etiology and epidemiology of Pythium root rot in hydroponic crops: current knowledge and perspectives retrieved http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0100-54052006000400001