Dishing the dirt

C&I Issue 9, 2014

Of the billions of microbes – bacteria, fungi, viruses, protozoa etc – in a spoonful of soil, many are neutral and do not affect plant growth, some are harmful pathogens, and some have a beneficial impact on plant health. The latter can act in various ways: by attacking harmful pests; preventing pathogen ingress into the plant; solubilising fertiliser and increasing nutrient uptake; and encouraging root growth.

Microbial biopesticides – particularly strains of Trichoderma fungi, Bacillus subtilis, and B. thuringiensis bacteria – have been commercially available for many years. More recently, as researchers have gained greater insight into plant–microbe interactions, biofertilisers and biostimulants, which improve the general health and growth of plants, have been introduced to the market.

In fact, the global market for agricultural biologics is $3bn, with biofertilisers and biostimulants accounting for approximately 50%, and biopesticides/bioinsecticides and biologic feed additives each accounting for 25%, according to Brian McSpadden-Gardener of The Ohio State University, US.

Plant–microbe interactions

One indication that microbes and plants interact is the much higher levels of microorganisms found around plant roots – the rhizosphere – compared with those in general soil. Plant growth-promoting rhizobacteria (PGPR), which include diverse strains of Bacillus, Streptomyces, Pseudomonas, Burkholderia and Agrobacterium, have attracted significant attention because they can affect plant growth directly and indirectly at all stages.

PGPRs can stimulate plant growth in various ways: via the fixation of atmospheric nitrogen; the synthesis of compounds that solubilise and sequester iron (siderophores) and other nutrients from the soil; and by generating plant hormones – eg salicylic acid, jasmonic acid, indole-3-acetic acid etc – and enzymes that affect plant processes. They can also indirectly improve plant growth by competing for resources that plant pathogens require and by producing antibiotics, eg 2,4-diacetylphloroglucenol (DAPG), that inhibit pathogen success.

PRPGs that express the enzyme 1-aminocyclopropane-1-carboxylate (ACC) deaminase, for example, are highly beneficial to plants, according to Bernard Glick from the University of Waterloo in Canada. This enzyme inactivates a precursor for the production of ethylene, which under normal conditions is involved in plant growth, but when produced in response to stress significantly impedes growth, leading to reduced productivity. ‘By preventing ethylene formation as a response to various stresses – drought, flooding, excess salt and heavy metals, and pathogen attack – productivity can be maintained,’ Glick says.

Jurriaan Ton of the University of Sheffield, UK, has shown that β-aminobutyric acid (BABA), a compound found in certain organisms, can boost broad-spectrum disease resistance. ‘BABA has long been recognised for its ability to induce long-lasting and broad-spectrum protection against pests and diseases. Our recent discovery of the receptor of BABA opens new exciting opportunities to exploit the benefits of this plant immune response. For instance, we are currently investigating how this receptor protein can be altered biochemically so that it remains in a constantly primed state without having to apply the chemical itself,’ he notes.

There are also bacteria, such as pink-pigmented facultative methylotrophs (PPFMs) from the genus Methylobacterium, which are distributed on plant surfaces and stimulate growth by supplying plant growth regulators, vitamins and some enzymes, according to Mark Holland of Salisbury University, Maryland, US. His group has also demonstrated that PPFMs are critical to seed germination and growth. ‘These bacteria produce the plant growth regulators cytokinin, gibberellin and auxin in metabolically meaningful amounts, and may also produce precursors to ethylene, abscisic acid and jasmonic acid,’ Holland notes.

Furthermore, adding PPFM bacteria – specifically mutants selected for overproduction of desired metabolites – to growing crops can increase growth and yield. As an example, Holland’s group has increased the methionine content of soy and added vitamin B12 to salad greens. PPFM bacteria, through their production of plant growth regulators, can also alter plant fertility, and could therefore be used for creating hybrids of many important crop plants, according to Holland. PPFM technology has been licensed by NewLeaf Symbiotics, based in St Louis, US.

Meanwhile, McSpadden-Gardener’s team has demonstrated that some bacterial strains can have multiple effects on plants. For example, certain Pseudomonas bacteria can produce a molecule that inhibits pathogens; stimulates secondary root growth; and induces plant host defences. Furthermore, the same strains can help crop plants tolerate acid soil stress and thus increase the crop yield. ‘These multiple modes of action make microbial inoculants particularly attractive as inputs,’ he says. Such strains are now on the market in products produced by 3Bar Biologics. ‘With the rise of new molecular techniques streamlining the discovery process, new strains with new activities are in the pipeline, so growers can expect to see more options in the future,’ McSpadden-Gardener adds.

‘Plants are incredibly versatile in anticipating and responding to harmful organisms in their environment,’ states Ton. Experiments done in his lab on root exudates of maize have shown that plants secrete specific root chemicals that recruit disease-suppressing microbes or emit herbivore-induced volatiles that prime the immune system of neighbouring plants. ‘In addition’, he says, ‘plants can increase the responsiveness of their immune systems and pass this trait on to their offspring through complex epigenetic mechanisms. Epigenetic mechanisms, for example, DNA methylation, result in inheritable functional changes to the genome without any change in the DNA sequence. As a result, these plants can “prepare” their progeny for longer-lasting biotic stresses in their environment that could potentially be threats.’

If Ton’s lab results translate to field conditions, he believes it will be possible to breed cereal varieties that are better able to recruit protective rhizosphere communities, thereby requiring fewer chemicals to control soil-borne diseases, which are particularly difficult to control using chemical pesticides.

Ton is excited about the idea of epigenetic transmission of plant defence traits. ‘Almost every month, there is new evidence from the literature suggesting that complex plant traits are partially controlled by heritable epigenetic traits. Breeding companies have already begun to extend their conventional selection programmes with epigenetic mapping populations that differ in patterns of DNA methylation but are genetically identical to select for desirable traits,’ he says. In Europe, where public opposition to genetic modification (GM) technology remains high, Ton suggests that this ‘epi-GM technology’ might be a more acceptable way forward for improving disease resistance and boosting the sustainability of crop production.

Many of the chemicals involved in plant signalling and growth that have been characterised over the past 10 years are volatile ‘green’ chemicals, according to Harsh Bais of the University of Delaware, US. His group has shown that when Arabidopsis thaliana plants are stressed, for example, by a bacterial attack, L-malic acid (MA) is secreted from their roots, selectively signalling and recruiting the beneficial rhizobacterium B. subtilis FB17, which then binds to the roots and forms a biofilm.

Bais’ research has revealed that root colonisation of FB17 can protect leafy greens – lettuce, spinach etc – from ingress by human pathogens that typically invade plants through stomata on leaf surfaces. The presence of FB17 in the rhizosphere results in the production of abscisic acid (ABA) and salicylic acid (SA), which are involved in signalling pathways that lead to closure of the guard cells of the stomata. He also notes that FB17 root colonisation can lead to improved drought resistance, because closure of the guard cells also helps reduce the loss of water.

The University of Delaware has licensed the FB17 technology to an undisclosed company for commercial development.

Growing business

Many companies are involved in the development of agricultural biologics, including smaller startups and large agrochemical companies. ‘The need to secure food supply is a world-wide challenge that requires innovative solutions to improve sustainability in food production,’ says Ton. Marcus Meadows-Smith, ceo of BioConsortia, US, a company with a proprietary method for selecting beneficial microbial consortia for crop improvement adds: ‘To feed the growing world population, we will need to double food production by 2050, and that cannot be achieved by doubling the land used for farming; it must be accomplished by improving productivity.’

Agricultural biologics have the potential to address this issue. It is important to realise, however, that just having a B. subtilis or Trichoderma species in a formulation does not mean the product will provide a beneficial response. Matthew S. Krause, product development manager with BioWorks, a US company that provides biological disease and pest management solutions to speciality agricultural industries, explains: ‘Only certain strains of microbes interact in a positive manner with plants; it takes extensive research and screening to identify those that have the biggest impact.’ BioWorks’ Trichoderma T-22 strain was found and developed commercially only after studying over 1000 different isolates,’ he explains.

In addition, a variety of different species in the soil is required to achieve optimum plant growth and protection. ‘Different bacteria are effective for solubilising fertilisers, increasing root growth, defending against pests and pathogens, and stimulating other plant activity,’ Meadows-Smith points out.

Most often, however, microbes are evaluated individually and then stacked together to formulate a product. BioConsortia, on the other hand, identifies effective consortia of bacteria that are naturally selected by a plant and that provide optimum plant traits/specific phenotypes. The company screens different soils under controlled conditions, selecting the top-performing plants – highest yields, sugar content etc – and identifying the naturally occurring consortia of microbes – both bacteria and fungi – present inside and around every part of the plant.

Currently, BioConsortia is conducting field trials in New Zealand and evaluating US soils in the laboratory for corn, soya beans, wheat and rice, though the technology is also being applied to major fruit and vegetable crops. The company’s pipeline includes microbial consortia for increased fertiliser utilisation and substitution of N, P, K and S; higher harvest yields, root mass and accelerated development etc. Most products will be seed treatments, but the firm will also produce some soil additives. ‘Seed treatments are ideal because they enable the microbes to grow along with the plants from the point of germination,’   says Meadows-Smith.

There are several factors to consider when developing agricultural biologics. ‘The microbes and the formulated product must be very robust and capable of providing optimum performance, and therefore all aspects of the planting conditions and the type of application must be considered. In addition, it is critical to ensure that the microbes adhere to the seed or foliage,’ comments Eda Reinot, director of R&D for BASF’s Functional Crop Care business. BASF has developed advanced polymer technologies that enable biologic treatments to adhere to seeds and plants without affecting the normal functioning of the seeds or plants or the performance of the beneficial microbes.

Appropriate use of agricultural biologics is also critical for their success. ‘Farmers must be very engaged in plant management in order to receive the optimum benefits of these products. They must know which microbes will be best for each crop and the growing environment, the optimum time to inoculate and the ideal conditions for optimum microbe growth,’ says McSpadden-Gardener. ‘Over-simplified, blanket approaches like those traditionally used with classical agrochemicals lead to lost opportunities to manage the variation that exists. Maximum success will depend on more thoughtful integration of microbials into different cropping systems,’ he asserts.

Juergen Huff, senior vp and head of BASF’s Functional Crop Care business, stresses that farming today is a high-technology industry, and that farmers are highly educated professionals looking for cost-effective solutions that will improve sustainable production, regardless of whether the product is chemical or biological in nature.

‘There is no one type of product that can provide a complete solution to achieving the dramatic productivity increases that are needed,’ states Huff. ‘A comprehensive approach is necessary, and therefore BASF develops biological and chemical products that are designed to be both compatible and complementary, are subjected to our stewardship review process and meet the increasingly strict regulatory requirements being implemented around the globe.’

BASF is also working to improve the consistency of its biologic products and further optimise their performance. Currently, the company offers biological products for use as seed treatments, foliar applications, and soil amendments for all stages of plant growth, including biofungicides, bioinsecticides, and plant nutrient improvements. For example, its Vault and Vault HP bacterial inoculants aid nitrogen fixation, while Vault HP plus Integral is a biological seed treatment system for soybeans consisting of multi-strain Bradyrhizobium japonicum species that stimulate root growth and nodulation and suppress fungal diseases for improved yields.

The R&D process for an agricultural biologic is extensive and expensive. Meadows-Smith notes for global products the cost for research, full field development and registration of a biopesticide is $20–50m and take five to six years, but that is still much less than required for a new synthetic pesticide, which costs >$200m and takes 8–10 years. He adds that biostimulants typically cost $10–20m and take three to five years.

Cynthia Challener is a freelance science writer based in Vermont, US

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