Diversity and biological control of Sclerotium rolfsii, causal agent of stem rot of groundnut

Groundnut (Arachis hypogaea L.) is an economically important legume crop in Vietnam and many other countries worldwide. Stem rot disease, caused by the soil-borne fungus Sclerotium rolfsii Sacc., is a major yield limiting factor in groundnut cultivation. Current control methods mostly rely on the extensive use of fungicides and on cultural practices. Both methods are not always effective and repeated fungicide use can lead to resistance development in the pathogen population. To further improve disease control and to reduce the use of chemical pesticides in groundnut cultivation, the feasibility of biological control of stem rot was investigated. The project initially focused on evaluating the occurrence and severity of stem rot disease in farmer fields in central Vietnam. It appeared that 5-25% of the groundnut plants in farmer fields in central Vietnam were infected by S. rolfsii. From infected fields, S. rolfsii isolates were collected and their genetic diversity was investigated as well as the variation in sensitivity to tebuconazole, a fungicide commonly used to control stem rot disease. Based on ITS-rDNA sequence analyses, three distinct groups were identified among a total of 103 randomly selected S. rolfsii field isolates, with the majority (n=90) in one ITS group. S. rolfsiiisolates originating from groundnut, tomato and taro were all pathogenic on groundnut and relatively sensitive to tebuconazole. However, the isolates displayed substantial diversity in various genetic and phenotypic traits, including mycelial compatibility, growth rate, and sclerotial characteristics. Subsequently, the efficacy of various beneficial bacteria to suppress stem rot disease was investigated. First, the biocontrol activity of well-characterized antagonistic Pseudomonas strains was evaluated in vitro and in growth chamber, nethouse and field experiments. Secondly, indigenous groundnut-associated bacteria were isolated, their diversity was investigated and their antifungal activity was analysed in nethouse and field experiments in central Vietnam. Finally, endophytic bacteria living inside groundnut nodules were isolated, characterized and tested for biocontrol and plant growth promotion under field conditions. For several of the most promising bacterial strains, the mechanisms involved in biocontrol of stem rot disease were investigated. The experiments conducted with well-characterized Pseudomonas strains showed that only phenazine-producing Pseudomonas chlororaphis strain Phz24 and Pseudomonas sp. strain SH-C52, producing the chlorinated lipopeptide thanamycin, inhibited hyphal growth of S. rolfsii and suppressed stem rot disease. Mutants of these strains that were deficient in phenazine or thanamycin production did not show any activity against S. rolfsii, indicating that these metabolites play an important role in suppression of stem rot disease. Other Pseudomonas strains producing structurally different lipopeptides did not or only marginally inhibit growth of S. rolfsii. These results suggested variation in sensitivity of the stem rot pathogen S. rolfsii for structurally different lipopeptides produced by Pseudomonas. Indigenous groundnut-associated bacteria with activity against S. rolfsii were classified by 16S-rDNA sequence analysis as γ-Proteobacteria (Pseudomonas), Bacteroidetes (Chryseobacterium), and Firmicutes (Bacillus). Among these indigenous bacterial genera, Pseudomonas sp. R4D2 consistently reduced stem rot disease under nethouse and field conditions. The genes and bioactive compounds involved in the biocontrol activity of strain R4D2 have not been identified yet, but phenotypic analyses suggest that biosurfactant production plays a central role. Chryseobacterium sp. R4B3 did not reduce stem rot disease in nethouse experiments, but was effective in suppression of stem rot disease under field conditions. Finally, the two indigenous isolates that were identified as Bacillus spp. did not reduce stem rot disease in nethouse experiments, but were effective in disease control under field conditions. Furthermore, both Bacillus strains significantly increased pod yield with dry weight increases of up to 30% relative to the control. Endophytic bacteria of groundnut nodules appeared to be genetically diverse. The majority was closely related to Sphingomonas while others were classified as Rhizobium, Burkholderia, Stenotrophomonas, and Sphingobacterium species. In nethouse experiments, occasionally, nodule formation on roots was observed on groundnut plants grown in soil supplemented with some of these endophytic isolates, but further analyses are required to establish that these isolates are indeed responsible for nodule formation and nitrogen fixation. For two of the identified endophytic isolates, i.e. Rhizobium sp. HR9 and Sphingomonas sp. HR32, field experiments showed that both strains were not effective against stem rot and other soil-borne diseases of groundnut, but significantly reduced leaf spot diseases caused by Cercospora and Cercosporidium spp. Application of these two endophytic bacterial strains also led to increases in pod yield of groundnut under field conditions. In conclusion, several of the bacterial strains tested in this study, in particular Pseudomonas and Bacillus spp., significantly improved pod yield of groundnut by 10 to 30%. The consistency in pathogen control and yield increase observed in two independent field experiments at two different locations in two consecutive years, holds great promise for further development of these bacterial strains as key components of an integrated strategy to manage multiple diseases of groundnut and to improve yield.