Microbial Tools for Advancing the Management of Soil and Seedling Health in Cotton Production Systems

Abstract

Soilborne plant filamentous fungal pathogens 'Thielaviopsis basicola (T. basicola)', 'Verticillium dahliae (V. dahliae)', and 'Fusarium oxysporum (F. oxysporum)' have been increasing steadily in Australian cotton, and an array of environmental and biological challenges limit their control. This study investigated the distribution and diversity of indigenous biocontrol agents and biofertilisers isolated from cotton production soils and their potential for controlling these pathogens. This work revealed that the Australian 'T. basicola' population had identical Internal Transcribed Spacer (ITS) sequences and a high level of morphological and pathogenic diversity with no apparent correlation between these traits. Polymerase Chain Reaction (PCR) amplification, sequencing and sequence alignment analysis of 26 'T. basicola' isolates found no change in ITS sequences which suggests that either the isolates are clonal decedents from a single strain or these sequences are subject to concerted evolution. Pathogenicity level was determined by inoculating soil with 5000 endoconidia/ml, a rate that was shown to cause 50% disease for isolate BRIP40192. Pathogenicity ranged from a high of 78% disease lesions caused by isolate PP231 to a moderate level of 36% lesions by isolate PP235. No correlation of pathogenicity with geographic collection site was established. High diversity in both pathogenicity and morphology indicated possible high mutation rates within the 'T. basicola' cotton population. A novel isolation method was developed and used to isolate 'T. basicola' suppressive microorganisms (bacteria and fungi) directly from soil samples. Four fungal pathogens were used to assess the isolation procedure including 'Thielaviopsis basicola', 'Verticillium dahliae', 'Verticillium fungicola (V. fungicola)' and 'Fusarium oxysporum'. The designed method showed great potential for the isolation of a wide variety of antagonistic microorganisms against a wide range of fungal pathogens. Further, the method was used to isolate potential biocontrol agents against 'T. basicola' from vastly different soils and 100% of these were confirmed to suppress the pathogen. Identification of isolates revealed a high level of diversity, with many belonging to groups already widely studied and used as biocontrol agents. This endorses the power of this technique to rapidly and directly isolate soilborne biocontrol microbial agents with the potential for inoculant development. A diverse range of potential biocontrol microorganisms is available in Australian cotton growing soils. Over 440 'T. basicola' suppressive fungal isolates, 63 'T. basicola' suppressive bacterial isolates and 61 'Trichoderma' isolates were screened in vitro using confrontation assays and root dipping pathogenicity assays. Most of the disease suppressive fungi (DSF) and all 'Trichoderma' isolates were found to inhibit the hyphal growth of the fungal pathogens 'T. basicola', 'V. dahliae' and 'F. oxysporum' and 100% of bacterial isolates suppressed 'T. basicola'. For 80 'T. basicola' suppressive fungal isolates plate based assays for growth rate, phosphate solubilisation and protease production were conducted and for 'Trichoderma' isolates chitinase, protease and indole-3-acetic acid (IAA) production colourimetric assays were conducted. 'T. basicola' suppressive fungal isolates and 'Trichoderma' isolates were found to possess multiple traits. Soil was co-inoculated with 'T. basicola' and isolates in order to assess biocontrol potential. All DSF including all 'Trichoderma' isolates assayed supressed black root rot within the soil and 93% of bacterial isolates also achieved some suppression. The most successful fungal isolate was a 'Penicillium' sp isolate APT30 which reduced 'T. basicola' lesions from 50% to 22%, while the most successful bacterial isolate was 'Bacillus amyloliquefaciens' isolate CNT24, reducing lesions to 26%. The 'Trichoderma viride' isolate BSTr1 reduced lesions to 20% and was the most successful of the 'Trichoderma' isolates. Bacterial populations possessing plant growth promoting traits were significantly different between fields, and results suggested that the addition of organic matter may influence the composition and activity of populations. Soil dilutions and plate based assays with selective phosphate solubilising, nitrogen fixing, ammonia oxidising and nitrifying media were used to determine bacterial soil populations, which were compared to cropping practices and soil analyses. To determine trait possession, phosphate solubilising bacteria from five different soils were individually replicated onto selective media. Multiple traits were common: 12-26% fixed nitrogen, 67-83% carried out ammonification, 54-89% oxidised ammonia and 32-48% produced IAA. Ten isolates with multiple growth promoting traits were subject to soil assays and nine were found to promote growth in one or both soils tested. This study demonstrated that a wealth of biocontrol and biofertiliser agents exist amongst the indigenous microflora of Australian soils and further application of the methodology developed here will yield additional candidates. Progress has been made towards the sustainable management of soilborne fungal diseases in the cotton agroecosystem, although further research is required for development and application of biocontrol agents.