Investigation of molecular and cellular aspects of cotton black root rot disease and the potential for biological control

Abstract

<p>The worldwide demand for high quality cotton has increased with time, and cotton production is now one of the most important rural industries in Australia. However, cotton growers face significant challenges in dealing with diseases that impact on crop yields. One of the most important diseases impacting cotton producers in Australia is black root rot disease, caused by the pathogenic fungi <em>Berkeleyomyces</em> spp. While a number of management practices have been suggested to help control this pathogen, there is not currently a complete solution to this problem. The main aims of this study were to gain a better understanding of the factors that affect disease severity, and explore the use of the bacterium <em>Azospirillum brasilense</em> as a biological control option for black root rot disease in cotton.</p><p> The distribution and abundance of <em>Berkeleyomyces</em> spp. in different soil samples from cotton farms (collected prior to planting) was investigated using quantitative polymerase chain reaction (qPCR). The incidence of the pathogen was higher in organically fertilised cotton fields compared to fields treated with mineral fertiliser. The relationship between crop rotation and pathogen abundance was also examined. Crop rotation with wheat and mung bean did not show any decrease in the soil load of <em>Berkeleyomyces</em> spp. However, soil properties, including iron and manganese levels, appeared to impact on the abundance of <em>Berkeleyomyces</em> spp. in soils.</p><p> The relationship between cotton cultivar, temperature and disease severity was also investigated, using <em>Berkeleyomyces rouxiae</em> BRIP40192 (a cotton isolate) and five cotton cultivars (Sicot 730, and the genetically modified cultivars Sicot 74BRF, Sicot 71BRF, Sicot 43BRF and Sicot 714B3F). Temperature had an influence on the proportion of root affected by black root rot lesions, and the water content of shoots, supporting the findings of previous studies that indicate temperature is a significant factor impacting disease severity and, consequently, plant growth. At higher temperatures (25°C) soil collected from the rhizosphere for all genetic modified cultivars (Sicot 74BRF, Sicot 71BRF, Sicot 43BRF and Sicot 714B3F) showed a significant decrease in the levels of <em>B. rouxiae</em> BRIP40192 recovered, compared to recovery at lower temperature (20°C).</p><p> To better understand the processes involved in disease, <em>in vitro</em> pathogenicity testing was carried out to examine the ability of <em>B. rouxiae</em> BRIP40192 to perceive and respond to signalling molecules produced by the roots of cotton seedlings. The results demonstrated that cotton root exudates stimulate the directional growth of <em>B. rouxiae</em>, although the strength of this attraction varied between cotton cultivars. The inclusion of the bacterium <em>Azospirillum brasilense</em> in the test was able to prevent this directional growth and stop spore germination. This finding suggests that there may be a role for <em>A. brasilense</em> as a potential biological control agent for <em>B. rouxiae</em>. Interaction tests between <em>B. rouxiae</em> BRIP40192 and <em>A. brasilense</em> strains showed that that the two were not antagonistic to each other, although <em>A. brasilense</em> strains showed a high capability to interact with <em>B. rouxiae</em> BRIP40192 hyphae.</p><p> The interaction between <em>B. rouxiae</em> BRIP40192, <em>A. brasilense</em> and cotton was investigated, focusing on the role played by border cells of the cotton root. Cotton border cells of all cultivars tested failed to trap <em>B. rouxiae</em> BRIP40192, however, <em>A. brasilense</em> strains, proposed as a potential biocontrol organisms, showed a high level of interaction with border cells of the five cotton cultivars and with border cells from wheat (often used in crop rotation on cotton farms). The colonisation of border cells by <em>A. brasilense</em> may assist with the suppression of germination of <em>B. rouxiae</em> BRIP40192 spores and retard directional growth in the rhizosphere, thus protecting seedlings from infection.</p><p> The results of this study help improve understanding of factors, such as temperature, cotton cultivar and farm management practices, which influence the distribution and severity of black root rot disease in Australian cotton farms. The results of this study also suggest that <em>A. brasilense</em> could be considered as a potential biological control agent to block or suppress the growth of black root rot disease in cotton fields.</p>