Integrated control of Rhizoctonia diseases on bean and cabbage

Vietnam is the third world’s largest vegetable producing country in the world. Due to the intensification of cropping systems, vegetable producers confront with the challenges of managing fungal pathogens, such as Rhizoctonia solani. This fungus is distributed both in virgin and cultivated soils as mycelium and sclerotia. R. solani mainly attacks its hosts when they are in their juvenile stages, causing various diseases on a wide range of economically important crops. Because resistant varieties are not available and cultural control is not sufficient, existing strategies to control Rhizoctonia diseases in Vietnam rely heavily on chemical pesticides. However, repeated pesticide applications may result in resistance development in the pathogen population, environmental pollution and negative health impacts. Hence, there is great interest in developing an integrated, sustainable strategy to control R. solani on vegetables cultivated in Vietnam. Before developing a control strategy it is necessary to know about the occurrence and distribution of Rhizoctonia spp. in Vietnam. Isolations from infested plants collected from Brassica fields showed that Brassica vegetables can be attacked by different anastomosis groups (AGs) of Rhizoctonia spp. Ninety three out of 97 Rhizoctonia isolates collected had multinucleate hyphal cells and were assigned to AG 1-IA, AG 1-ID, AG 1-IB, AG 1-IG, AG 4-HGI, AG 2-2 and AG 7. The other four isolates in our collection belonged to AG-A and AG-Fc of binucleate Rhizoctonia. To our best knowledge, this is the first time AG 1-IA and AG 1-ID are isolated from Brassica vegetables. Bioassays demonstrated significant divergence in pathogenicity among AGs, in which only isolates of AG 4-HGI, AG 2-2 and AG-Fc could induce severe disease symptoms under all conditions tested. Interestingly, we found that the occurrence of the different AGs are related to the cropping systems and cultural practices, implying that agricultural practices might determine the AGs associated with Brassica crops in Vietnam. Pseudomonas sp. CMR12a is an antagonistic bacterium isolated from the roots of healthy cocoyam in diseased cocoyam fields in Cameroon. This strain excretes phenazines and two types of cyclic lipopeptides (CLPs), designated sessilin and orfamide. In this thesis, we showed that Pseudomonas sp. CMR12a successfully protected bean seedlings from disease and the biocontrol activity of this strain is attributed to an additive effect of phenazines and sessilin. Subsequent in vivo experiments conducted with the same bacterial strain and R. solani AG 2-2 in three different potting soil/sand mixtures revealed that biocontrol activity of phenazines and CLP sessilin was substrate-dependent. The proportion of sand present in substrates positively correlated with the spreading rate of Rhizoctonia hyphae and the disease severity. In substrates containing 50% or 75% of potting soil, the production of phenazines or sessilin was sufficient to suppress bean root rot. However, in sand-dominant substrate, the presence of both phenazines and sessilin was needed to be effective, possibly due to the high disease pressure. In a further stage of the research, we were interested in exploring the ability to induce systemic resistance against web blight on bean by CMR12a. The application of CMR12a to growth substrate significantly reduced disease caused by R. solani AG 2-2 on leaves of seedlings. In order to test whether phenazines, sessilin and orfamide are involved in CMR12a-mediated ISR, mutants deficient in the production of phenazines and/or CLPs production were included in the experiments. Mutants disrupted in sessilin or orfamide biosynthesis were as effective as the parental strain. However, two mutants deficient in the production of both sessilin and orfamide completely lost their disease suppressive effect. These results imply that sessilin and orfamide are the determinants for induced resistance in wild type Pseudomonas sp. CMR12a. In addition to controlling Rhizoctonia diseases on bean, Pseudomonas sp. CMR12a also showed its biocontrol capacity towards the germination of Rhizoctonia sclerotia and the severity of damping-off disease on cabbage. The results of plant experiments and microplate assays indicated that the presence of phenazines or the coexistence of sessilin and orfamide reduced sclerotia viability in vitro and suppressed disease development in vivo. These observations suggest that there is a synergistic effect of sessilin and orfamide in the control of R. solani. The synergistic interaction between these CLPs was confimed by microscopic analyses showing that the combination of a double phenazine- and orfamide-negative mutant and a double phenazine- and sessilin-negative mutant or purified orfamide significantly reduced mycelial growth of Rhizoctonia and increased branching of hyphal tips. Data obtained also implicate the dose-dependent direct antagonistic effect of orfamide in vitro and the essential role of sessilin in root colonization in planta. In the next part of this work, we focused on the potential of coir pith, a side product abundantly available in Vietnam, to reduce the persistence of Rhizoctonia sclerotia and to suppress the severity of damping-off disease. Pot trials showed that sclerotial mortality was 78% when coir pith was added at a rate of 5% (w/w). A reduction in sclerotia viability was observed together with an increase in the number of sclerotia infected with parasitic fungi and the raise in the population density of Trichoderma spp. and fluorescent pseudomonads in soil. Based on these results it was hypothesized in accordance to previous studies that the incorporation of lignin favored the growth of lignin-degrading fungi. Some extracellular enzymes such as lignin peroxidase, manganese peroxidase and laccase produced by lignin degraders can affect melanin. Sclerotia with degraded melanin are more susceptible to antagonists such as Trichoderma spp., Actinomycetes and Gram negative bacteria. The decrease in sclerotia viability due to coir pith incorporation was related with a significant decrease in the severity of damping-off disease. Moreover, the disease suppressive ability could be enhanced when coir pith was combined with Pseudomonas sp. CMR12a, possibly due to the biocontrol effect of CMR12a