Ecology of Microbes in Cotton Soils

Peter McGee, Jenny Saleeba, David Midgley, Alice Simpson, Stella Loke, Leonie Whiffen, Sam Alomari, Zoe Commandeur, Endymion Cooper. School of Biological Sciences University of Sydney

Microbes play many important roles in soil. Among the functions are decomposition of organic matter, cycling of minerals, plant uptake of minerals, plant pathology, degradation of organic toxins, sequestration of metals, and suppression of deleterious organisms, including other microbes. Microbes also influence soil structure through release of ‘glues’ that cement fine particles in to aggregates, and fungal hyphae physically entangle soil aggregates into larger aggregates. The role of microbes is strongly influenced by presence of plants because roots release ‘food’ for many microbes, and indeed, we think of the root/mycorrhiza symbiosis as the predominant source of energy for bacteria, with the release of sloughed cells, cell wall materials and more complex compounds as the main nutrients for saprotrophic fungi. Arthropods eat various microbes and organic matter, and are themselves, dead or alive, the diet of other organisms. Microbes and plants are intimately linked, each influencing the function of the other. Until recently, studies of the functions of microbes in soil have been limited in extent and usefulness because of the complexity of the microbial community.

Our understanding of microbes in cotton soils is limited. Significant research has been developed in understanding some pathogens, and we are developing clearer understanding of the ecology and function of arbuscular mycorrhizal (AM formerly known as VAM) fungi. We can also borrow from extensive research on, for instance, the role of microbes in cycling of nitrogen. Otherwise, we lack an appreciation of what is happening to the communities of microbes when we cultivate and grow crops including cotton. In addition, the impact of any changes in community on the growth and productivity of the cotton plant are unknown.

Our research group set out to examine three topics: we documented the diversity of common microbes in cotton soils and followed this with research targeting interactions between microbes and seedling pathogens, we examined the impact of cultivation of cotton on diversity and abundance of AM fungi, and we commenced a study to determine the importance of microbes in sequestering carbon in soil.

More than 10,000 microbes might be found in one gram of dry soil. These communities do not change significantly over short distances in soil. In other words, an extremely high diversity and abundance of microbes is present in soil. The abundance of different types of microbe, however, is extremely skewed. A few microbes are common, and most of the rest are uncommon. Microbial communities are also dynamic. The relative abundance of a species can change dramatically and rapidly if the conditions change to favour or disadvantage one or other microbe. Our approach was to compare the communities of microbes under cotton with communities in similar soils but under native vegetation, or under other vegetation regimes, or different soil types, or different rotations. We used a comparative approach in case important groups are missing, or present, in cotton soils. Because so few microbes can be cultured, we mainly used molecular techniques. Molecular techniques rely on extraction of representative DNA from soil, with subsequent replication of specific sequences that can then be matched to a data base of sequences to indicate an identity. Thus common microbes will be readily identified and new or unusual microbes will remain unknown or poorly identified. Molecular techniques also select for particular types of DNA. The answers using DNA techniques, while incomplete, add to information gained from culture based techniques. In addition, we used culture based techniques that specifically targeted groups we wished to study and these studies were carried out over a number of years.

The diversity studies indicated that communities of microbes in cotton soils are reduced, and remarkably similar to one another, when compared to native soils. The soil type and rotation did not seem to have a major impact on the community in cultivated soil (Fig 1 and 2). These data are preliminary. We are comparing communities of microbes under cotton with other cropping systems, managements and climatic zones. Reduced diversity has been reported by others, is consistent with our culture based results, and was expected. The similar community structure under different crops and rotations and in soil types was unexpected. A similar community structure enables data from one soil type to be applied to another with some confidence.

Ascomycete diversity

150

125

100

75 TRFs

50

25

0 BNR CNR WAR JC ADJ CW FF BF Soils

Figure 1. Diversity of Ascomycetes (number of TRFs) detected in soils from under native (BNR, CNR, WAR and JC) or cultivated (ADJ, CW, FF, BF) soils.

Microbial communities in soil under native vegetation were highly variable. Where the vegetation was grassland, the microbial communities were somewhere between those of crop and native shrub and woodland. In one extreme situation from another project, we detected a tiny quantity of DNA in one sample of exposed spoil from Woods Reef mine site. We detected some groups of microbes (presumably common) in the native soils and failed to detect them in cropping soil. Indeed, some groups were readily detected in cropping soil and were less common under native vegetation. Again, our culture based methods resulted in similar overall differences, but the specific groups differed.

Basidiomycete diversity

140

120

100

80

TRFs 60

40

20

0 BNR CNR WAR JC ADJ CW FF BF Soils

Figure 2. Diversity of Basidiomyctes (number of TRFs) detected in soils from under native (BNR, CNR, WAR and JC) or cultivated (ADJ, CW, FF, BF) soils.

Two important groups were commonly detected by molecular methods in native soils: bacteria within the Actinobacteria, and fungi in the Trichocomaceae (in bold in Table 1) were common in native soils, and not detected or were rare in cropping soils. Both groups of microbes are renowned for their production of antibiotics. Thus they are possibly important for inhibiting other organisms in soil. In addition, some of the fungi are associated with solubilisation of P from recalcitrant sources. This result indicates that natural microbial defenses of a soil might be reduced under cropping, and that some minerals might be less available.

The microbes that were common in cropping soils included groups related to or within either Fusarium (in bold in Table 1) or Rhizoctonia. Both groups are known as minor or major pathogens of many crop plants. To summarise, then, cropping soils have many microbes that may be pathogens, and native soils have microbes that are associated with inhibition of other organisms. What happens when you put the inhibitory microbes into cropping soils containing pathogens is the focus of our current project.

Table 1. Selected common Ascomycetes detected in cotton-wheat (CW) rotation and soils from under nearby native vegetation (BNR and CNR) of the Narrabri area. Note the presence of Fusarium in cropping soil and Trichocomaceae in native soil.

SITE Closest ID Tentative ID CW Humicola fuscoatra Ascomycete #1 CW Fusarium lateritium Hypocreomycetidae taxon #1 CW Cordyceps jezoensis Hypocreales taxon #1 CW Microdochium sp. Xylariales taxon #1 CW Myrothecium cinctum Hypocreales taxon #4 CW Leaf litter ascomycete Xylariales taxon #2 CW Fusarium oxysporum Hypocreales, Fusarium sp. #1

BNR Bisporella citrina Pezizomycotina taxon #1 BNR Cordyceps sp. Ascomycete #2 BNR Phaeococcomyces chersonesos Chaetothyriales taxon #1 BNR Hortaea werneckii Dothideales taxon #1 BNR Ericoid mycorrhizal sp. Helotiales taxon #1 BNR Phaeococcomyces chersonesos Chaetothyriales taxon #2 BNR Phoma sp. 2 Phoma-like ascomycete #1 BNR Penicillium pimiteouiense Trichocomaceae taxon #1 BNR Penicillium striatisporum Trichocomaceae – Penicillium sp. #1

CNR Neosartorya stramenia Trichocomaceae taxon #2 CNR Coniosporium sp. Ascomycete #3 CNR Botryosphaeria dothidea Ascomycete #4 CNR Didymella cucurbitacearum Dothideomycetes, Didymella sp. #1 CNR Didymella cucurbitacearum Dothideomycetes taxon #1 CNR Ericoid mycorrhizal sp. Helotiales taxon #2 CNR Penicillium striatisporum Trichocomaceae – Penicillium sp. #2 CNR Penicillium canescens Trichocomaceae – Penicillium sp. #3 CNR Massarina lacustris Dothideomycetes taxon #2 CNR Curvularia trifolii Pleosporales, Curvularia sp. #1

Stella Loke examined the impact of cropping on diversity of arbuscular mycorrhizal (AM) fungi. This project was based on culture based research indicating that a few species of fungi with small spores were common in cropping soil. Molecular research from the UK indicated that the diversity of AM fungi might be greater than we supposed. Research from the USA indicated that cropping with one plant species, such as back to back cotton might reduce diversity of AM fungi. We had also shown in previous research that growth of cotton seedlings was greater when seedlings were inoculated with many compared to one AM fungus. Thus we were concerned that if diversity was reduced plant growth and lint production might also be reduced. Stella used molecular techniques, and showed clearly that diversity of AM fungi was quite high in cotton soils, and that rotations did not suppress fungal diversity. Even more important was the observation that many types of AM fungi survived in the 7 year fallow maintained by David Nehl at ACRI. In other words, AM fungi are amazingly good survivors. Other work by David showed that the abundance of AM in roots of cotton recover rapidly following drought or fallow. It is important to note, though, that Stella confirmed research that indicates that different crops select the species of AM fungi that develop colonies in their roots. We know that each fungus has a different effect on plant uptake of phosphate, and each fungus interacts with its environment differently. Thus rotations will result in changing abundances in communities of AM fungi, with an unknown, impact on plant growth and productivity. At a gross level, changing dominance in communities of AM fungi is unlikely to cause a significant decline in lint production. However, we hope to develop further the question of what determines which species of AM fungus colonises cotton, and the effect of the competition between fungi on plant growth and development.

Research supported by the Cotton Industry during the mid 1990s showed that overall abundance of AM fungi was less under cotton than in complex perennial plant systems. Between 0.05 and 0.5 m of fungal hyphae per gram of soil were measured under cotton crops. Up to 4 m has been observed in native vegetation from northern NSW. Reasons for these differences are unknown. However, the lack of biomass of AM fungi is important, for two main reasons: AM fungi influence soil structure, and they constitute a significant fraction of the organic carbon in soil. AM fungi are associated with soil structure in two important ways. AM fungi release glomalin, a poorly characterised glycoprotein, into soil. Glomalin is thought to aggregate fine soil particles. In addition, hyphae physically enmesh aggregates. These macroaggregates enable movement of water and air through the soil profile. Thus abundance of AM fungi will influence plant health through the effect of the fungi on soil structure.

Cropping soils around the world have lost an alarming amount of carbon. Loss of carbon has local and global consequences. At a local level, decline in organic carbon is detrimental to the biological and physical properties of the soil making it less resistant and resilient to management practices. Globally, loss of carbon from soil used for agriculture accounts for the release of 1 - 2 b

tonnes of carbon in the form of CO2, contributing significantly to global warming. It is widely believed that anthropogenic global warming will result in reduced rainfall and more unpredictable changes in weather patterns for Australian cotton growing regions. Thus Leonie Whiffen set out to determine whether AM fungi contribute to long lived soil carbon and whether the contribution is significant enough for changes in management of AM fungi to be considered as a means to sequester carbon in soil.

Transect surveys of naturally vegetated and field soils demonstrate significantly lower concentrations of organic matter and glomalin related materials in cropping soil. Work to further characterise glomalin has shown that published methods of measuring glomalin also partially quantify the phenolic fraction from soil. The possible interaction between glomalin and phenolic acids is currently under investigation. A long term experiment to determine the contribution of AM fungi to stores of organic carbon in different soil types has now been completed and data is being analysed.

Our final comment is on the current project examining whether we can influence seedling disease by inoculating field soil with fungi in the Trichocomaceae. This research has two theoretical components. Research out of the USA indicates that high plant diversity results in greater total plant productivity, and that diversity of functional types of plants results in greater stability and productivity of the plant community. The relative importance of diversity of microbes on plant function has not been tested, but may be hypothesised from the second theoretical component concerning diversity of microbes and suppression of pathogens. Most biocontrol systems that use a single agent of control have failed in some soils or climates. In contrast, well composted mixed plant material with diverse microbial community suppresses pathogens of horticultural plants in pots. Thus we set out to determine whether amendment of cropping soil with a diversity of microbes, or a diversity of functions among the microbes would suppress seedling diseases of cotton. Suppression of disease may arise from inhibition of the seedling pathogens, induction of plant response and increased seedling nutrition. We have isolated fungi and bacteria from native and cropped soils, using selective media. Initial studies concentrated on fungi in the Trichocomaceae because members include some known to produce antibiotics, others to release P from recalcitrant sources, and others to degrade plant matter. Indeed, a few may cause disease under extreme conditions. In a preliminary experiment, we found that inoculation of cotton seedlings with up to 35 isolates of Trichocomaceae reduced disease caused by Thielaviopsis (Black Root Rot). When we added compost from the Sydney basin or soil from native vegetation, as sources of diverse and complex communities of microbes, disease was unaffected. We are still analysing the experiment, and have yet to determine whether P uptake was altered. In subsequent experiments, we will need to determine the mechanisms underlying this result, especially whether a single fungus or many fungi are the determinants of disease suppression. However, we feel encouraged to continue exploring this potential method of suppressing soil borne pathogens.

Overall, our research starts to clarify some of the complex interactions taking place in soil. We are contributing to both theoretical and practical ends. We have research that supports theories proposing diversity leading to resistance and resilience of ecosystems. We also have data that indicates some of the important functions of microbes may be harnessed to enable a more sustainable approach to producing food and fibre.