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

Cropping systems and cultural practices determine the Rhizoctonia anastomosis groups associated with Brassica spp. in Vietnam

Ninety seven Rhizoctonia isolates were collected from different Brassica species with typical Rhizoctonia symptoms in different provinces of Vietnam. The isolates were identified using staining of nuclei and sequencing of the rDNA-ITS barcoding gene. The majority of the isolates were multinucleate R. solani and four isolates were binucleate Rhizoctonia belonging to anastomosis groups (AGs) AG-A and a new subgroup of A-F that we introduce here as AG-Fc on the basis of differences in rDNA-ITS sequence. The most prevalent multinucleate AG was AG 1-IA (45.4% of isolates), followed by AG 1-ID (17.5%), AG 1-IB (13.4%), AG 4-HGI (12.4%), AG 2-2 (5.2%), AG 7 (1.0%) and an unknown AG related to AG 1-IA and AG 1-IE that we introduce here as AG 1-IG (1.0%) on the basis of differences in rDNA-ITS sequence. AG 1-IA and AG 1-ID have not been reported before on Brassica spp. Pathogenicity tests revealed that isolates from all AGs, except AG-A, induced symptoms on detached leaves of several cabbage species. In in vitro tests on white cabbage and Chinese cabbage, both hosts were severely infected by AG 1-IB, AG 2-2, AG 4-HGI, AG 1-IG and AG-Fc isolates, while under greenhouse conditions, only AG 4-HGI, AG 2-2 and AG-Fc isolates could cause severe disease symptoms. The occurrence of the different AGs seems to be correlated with the cropping systems and cultural practices in different sampling areas suggesting that agricultural practices determine the AGs associated with Brassica plants in Vietnam

Rice sheath rot : an emerging ubiquitous destructive disease complex

Around one century ago, a rice disease characterized mainly by rotting of sheaths was reported in Taiwan. The causal agent was identified as Acrocylihdrium oryzae, later known as Sarocladium oryzae. Since then it has become clear that various other organisms can cause similar disease symptoms, including Fusarium sp. and fluorescent pseudomonads. These organisms have in common that they produce a range of phytotoxins that induce necrosis in plants. The same agents also cause grain discoloration, chaffiness, and sterility and are all seed-transmitted. Rice sheath rot disease symptoms are found in all rice growing areas of the world. The disease is now getting momentum and is considered as an important emerging rice production threat. The disease can lead to variable yield losses, which can be as high as 85%. This review aims at improving our understanding of the disease etiology of rice sheath rot and mainly deals with the three most reported rice sheath rot pathogens: S. oryzae, the Fusarium fujikuroi complex, and Pseudomonas fuscovaginae. Causal agents, pathogenicity determinants, interactions among the various pathogens, epidemiology, geographical distribution, and control options will be discussed

Fluorescent Pseudomonas and cyclic lipopeptide diversity in the rhizosphere of cocoyam (Xanthosoma sagittifolium)

Cocoyam (Xanthosoma sagittifolium (L.)), an important tuber crop in the tropics, is severely affected by the cocoyam root rot disease (CRRD) caused by Pythium myriotylum. The white cocoyam genotype is very susceptible while the red cocoyam has some field tolerance to CRRD. Fluorescent Pseudomonas isolates obtained from the rhizosphere of healthy red and white cocoyams from three different fields in Cameroon were taxonomically characterized. The cocoyam rhizosphere was enriched with P. fluorescens complex and P. putida isolates independent of the plant genotype. LC-MS and NMR analyses revealed that 50% of the Pseudomonas isolates produced cyclic lipopeptides (CLPs) including entolysin, lokisin, WLIP, putisolvin and xantholysin together with eight novel CLPs. In general, CLP types were linked to specific taxonomic groups within the fluorescent pseudomonads. Representative CLP-producing bacteria showed effective control against CRRD while purified CLPs caused hyphal branching or hyphal leakage in P. myriotylum. The structure of cocoyamide A, a CLP which is predominantly produced by P. koreensis group isolates within the P. fluorescens complex is described. Compared with the white cocoyam, the red cocoyam rhizosphere appeared to support a more diverse CLP spectrum. It remains to be investigated whether this contributes to the field tolerance displayed by the red cocoyam

The involvement of phenazines and cyclic lipopeptide sessilin in biocontrol of Rhizoctonia root rot on bean (Phaseolus vulgaris) by Pseudomonas sp. CMR12a is influenced by substrate composition

Pseudomonas sp. CMR12a is an effective biocontrol agent that produces various antifungal metabolites including phenazine antibiotics and cyclic lipopeptides. In this study, we wanted to investigate the influence of substrate composition on the role of phenazines and the cyclic lipopeptide (CLP) sessilin in biocontrol of bean root rot caused by Rhizoctonia solani AG 2-2. Disease severity and spreading rate of R. solani were determined in three substrates, containing different ratios of potting soil and sand, inoculated with Pseudomonas sp. CMR12a or its mutants impaired in phenazine and/or sessilin biosynthesis. In the mixtures containing 50 or 75 % of potting soil, the presence of either phenazines or sessilin was sufficient to suppress bean root rot. However, in the mixture containing only 25 % of potting soil, the involvement of both compounds was required to provide better protection. The ratio between potting soil and sand also determined the spreading rate of Rhizoctonia hyphae. The mixture containing 75 % of potting soil was the most suppressive against R. solani invasion. The use of various potting soil/sand combinations affected the development of bean root rot disease but Pseudomonas sp. CMR12a effectively controlled the disease in all substrates. Depending on the disease pressure, however, the production of either phenazines or sessilin, or both compounds is required for effective biocontrol

Role of phenazines and cyclic lipopeptides produced by pseudomonas sp CMR12a in induced systemic resistance on rice and bean

Pseudomonas sp. CMR12a produces two different classes of cyclic lipopeptides (CLPs) (orfamides and sessilins), which all play a role in direct antagonism against soilborne pathogens. Here we show that Pseudomonas sp. CMR12a is also able to induce systemic resistance to Magnaporthe oryzae on rice and to the web blight pathogen Rhizoctonia solani AG2-2 on bean. Plant assays with biosynthesis mutants of Pseudomonas sp. CMR12a impaired in the production of phenazines and/or CLPs and purified metabolites revealed that distinct bacterial determinants are responsible for inducing systemic resistance in these two pathosystems. In rice, mutants impaired in phenazine production completely lost their ability to induce systemic resistance, while a soil drench with pure phenazine-1-carboxamide (PCN) at a concentration of 0.1 or 1 mu M was active in inducing resistance against M. oryzae. In bean, mutants that only produced phenazines, sessilins or orfamides were still able to induce systemic resistance against Rhizoctonia web blight, but a balanced production of these metabolites was needed. This study not only shows that Pseudomonas sp. CMR12a can protect rice to blast disease and bean to web blight disease, but also displays that the determinants involved in induced systemic resistance are plant, pathogen and concentration dependent

Comparative Study of Phosphorous-Acid-Containing Products for Managing Phytophthora Blight of Bell Pepper

Phytophthora blight of pepper caused by Phytophthora capsici is a major constraint to bell pepper (Capsicum annuum) production. The long-term effectiveness of chemicals currently in use against P. capsici is uncertain due to the development of fungicide resistance by this pathogen. Hence, the efficacy of alternative chemicals such as phosphorous-acid-containing products was evaluated in this study. In in vitro tests, ProPhyt, K-Phite, Lexx-A-Phos, Agri-Fos, and Nutri-Phite were less effective in inhibiting mycelial growth (EC50 = 50.5 to 324.4 µg mL−1) and sporangium formation (EC50 = 6.1 to 225.7 µg mL−1) of two P. capsici isolates, but more effective against zoospore germination compared with mefenoxam. Among phosphorous-acid-containing products tested, Nutri-Phite was most effective in inhibiting mycelial growth of both P. capsici isolates. In greenhouse studies, Nutri-Phite was effective against Phytophthora blight used as drench. The use of Nutri-Phite, Agri-Fos, ProPhyt, and K-Phite could induce systemic resistance against foliar blight when applied to the root and potting mix. The results indicated that some phosphorous-acid-containing products have the potential to lower disease occurrence and delay Phytophthora blight of bell pepper without phytotoxic effects. The utility of the systemic protection induced by these products is promising in Phytophthora blight management

Comparative Study of Phosphorous-Acid-Containing Products for Managing Phytophthora Blight of Bell Pepper

Phytophthora blight of pepper caused by Phytophthora capsici is a major constraint to bell pepper (Capsicum annuum) production. The long-term effectiveness of chemicals currently in use against P. capsici is uncertain due to the development of fungicide resistance by this pathogen. Hence, the efficacy of alternative chemicals such as phosphorous-acid-containing products was evaluated in this study. In in vitro tests, ProPhyt, K-Phite, Lexx-A-Phos, Agri-Fos, and Nutri-Phite were less effective in inhibiting mycelial growth (EC50 = 50.5 to 324.4 µg mL−1) and sporangium formation (EC50 = 6.1 to 225.7 µg mL−1) of two P. capsici isolates, but more effective against zoospore germination compared with mefenoxam. Among phosphorous-acid-containing products tested, Nutri-Phite was most effective in inhibiting mycelial growth of both P. capsici isolates. In greenhouse studies, Nutri-Phite was effective against Phytophthora blight used as drench. The use of Nutri-Phite, Agri-Fos, ProPhyt, and K-Phite could induce systemic resistance against foliar blight when applied to the root and potting mix. The results indicated that some phosphorous-acid-containing products have the potential to lower disease occurrence and delay Phytophthora blight of bell pepper without phytotoxic effects. The utility of the systemic protection induced by these products is promising in Phytophthora blight management

Interplay between orfamides, sessilins and phenazines in the control of Rhizoctonia diseases by Pseudomonas sp CMR12a

We investigated the role of phenazines and cyclic lipopeptides (CLPs) (orfamides and sessilins), antagonistic metabolites produced by Pseudomonas sp. CMR12a, in the biological control of damping-off disease on Chinese cabbage (Brassica chinensis) caused by Rhizoctonia solaniAG 2-1 and root rot disease on bean (Phaseolus vulgaris L.) caused by R. solaniAG 4-HGI. A Pseudomonas mutant that only produced phenazines suppressed damping-off disease on Chinese cabbage to the same extent as CMR12a, while its efficacy to reduce root rot on bean was strongly impaired. In both pathosystems, the phenazine mutant that produced both CLPs was equally effective, but mutants that produced only one CLP lost biocontrol activity. In vitro microscopic assays revealed that mutants that only produced sessilins or orfamides inhibited mycelial growth of R. solani when applied together, while they were ineffective on their own. Phenazine-1-carboxamide suppressed mycelial growth of R. solaniAG 2-1 but had no effect on AG 4-HGI. Orfamide B suppressed mycelial growth of both R. solani anastomosis groups in a dose-dependent way. Our results point to an additive interaction between both CLPs. Moreover, phenazines alone are sufficient to suppress Rhizoctonia disease on Chinese cabbage, while they need to work in tandem with the CLPs on bean

Pairwise sequence similarities of unknown isolates LDDL02-1 and DNBH05-1-2 to all known AGs from the curated database in Table S2 in File S2.

<p>LDDL02-1 shows most similarity to AG 6 and AG-Fb. DNBH05-1-2 shows highest pairwise sequence similarity to AG 1-IA and AG 1-IE.</p><p>Pairwise sequence similarities of unknown isolates LDDL02-1 and DNBH05-1-2 to all known AGs from the curated database in Table S2 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111750#pone.0111750.s002" target="_blank">File S2</a>.</p