Resident bacteria, nitric oxide emission and particle size modulate the effect of Brassica napus seed meal on disease incited by Rhizoctonia solani and Pythium spp

Amendment of orchard soil with low-glucosinolate Brassica napus (rape) seed meal (RSM) suppresses infection of apple roots by Rhizoctonia solani but increases incidence of Pythium spp. infection. Following incorporation of Brassica sp. seed meals, soils were monitored for changes in populations of selected saprophytic and plant pathogenic microorganisms. When conducted in pasteurized soil, which possessed high numbers of Bacillus spp. and lower than detectable numbers of Streptomyces spp., RSM amendment did not provide control of R. solani. Populations of streptomycetes in RSM-amended soil increased to stable levels >20-fold higher than in non-amended soil. Disease suppressiveness was restored to pasteurized RSM-amended soil by adding any of several Streptomyces strains. Maximal rates of nitrification in orchard soil, determined by nitric oxide emission, were observed within two weeks following RSM amendment and inhibition of nitrification via application of nitrapyrin abolished the capacity of RSM to suppress R. solani infection of apple roots when seedlings were planted one day after soil amendment. Apple seedling mortality and Pythium spp. root infection were highest for seedlings planted immediately following incorporation of B. napus cv. Athena RSM, particularly when meal was added in a flake rather than powder form. Lower infection frequencies were observed for seedlings planted four weeks after RSM incorporation, even for soil in which densities of culturable Pythium spp. had not declined. Our results demonstrate that suppression of Rhizoctonia root rot in response to RSM amendment requires the activity of the resident soil microbiota and that initial disease control is associated with the generation of nitric oxide through the process of nitrification.     

Mechanisms of natural soil suppressiveness to soilborne diseases

Suppressive soils are characterized by a very low level of disease development even though a virulent pathogen and susceptible host are present. Biotic and abiotic elements of the soil environment contribute to suppressiveness, however most defined systems have identified biological elements as primary factors in disease suppression. Many soils possess similarities with regard to microorganisms involved in disease suppression, while other attributes are unique to specific pathogen-suppressive soil systems. The organisms operative in pathogen suppression do so via diverse mechanisms including competition for nutrients, antibiosis and induction of host resistance. Non-pathogenic Fusarium spp. and fluorescent Pseudomonas spp. play a critical role in naturally occurring soils that are suppressive to Fusarium wilt. Suppression of take-all of wheat, caused by Gaeumannomyces graminis var. tritici, is induced in soil after continuous wheat monoculture and is attributed, in part, to selection of fluorescent pseudomonads with capacity to produce the antibiotic 2,4-diacetylphloroglucinol. Cultivation of orchard soils with specific wheat varieties induces suppressiveness to Rhizoctonia root rot of apple caused by Rhizoctonia solani AG 5. Wheat cultivars that stimulate disease suppression enhance populations of specific fluorescent pseudomonad genotypes with antagonistic activity toward this pathogen. Methods that transform resident microbial communities in a manner which induces natural soil suppressiveness have potential as components of environmentally sustainable systems for management of soilborne plant pathogens. This revised version was published online in August 2006 with corrections to the Cover Date.     

Biocontrol of Rhizoctonia solani damping-off of sugar beet with native Streptomyces strains under field conditions

A 3-year study was conducted to evaluate the effectiveness of two disease-suppressive Streptomyces spp. to control sugar beet Rhizoctonia solani damping off under field conditions. Streptomyces seed treatments reduced seedling damping off in naturally (2005) and artificially (2006 and 2007) infested soils. All biocontrol agents provided better efficacy than Vitavax to control seedling damping-off. There were no significant differences among Streptomyces isolates. Isolate C increased plant stand by 19.5, 50.5 and 53.75% in 2005, 2006 and 2007, respectively. Evaluation of final harvest revealed that the root yield of the biocontrol agents increased compared to untreated control in these years.     

Role of Bacterial Communities in the Natural Suppression of Rhizoctonia solani Bare Patch Disease of Wheat (Triticum aestivum L.)

Rhizoctonia bare patch and root rot disease of wheat, caused by Rhizoctonia solani AG-8, develops as distinct patches of stunted plants and limits the yield of direct-seeded (no-till) wheat in the Pacific Northwest of the United States. At the site of a long-term cropping systems study near Ritzville, WA, a decline in Rhizoctonia patch disease was observed over an 11-year period. Bacterial communities from bulk and rhizosphere soil of plants from inside the patches, outside the patches, and recovered patches were analyzed by using pyrosequencing with primers designed for 16S rRNA. Taxa in the class Acidobacteria and the genus Gemmatimonas were found at higher frequencies in the rhizosphere of healthy plants outside the patches than in that of diseased plants from inside the patches. Dyella and Acidobacteria subgroup Gp7 were found at higher frequencies in recovered patches. Chitinophaga, Pedobacter, Oxalobacteriaceae (Duganella and Massilia), and Chyseobacterium were found at higher frequencies in the rhizosphere of diseased plants from inside the patches. For selected taxa, trends were validated by quantitative PCR (qPCR), and observed shifts of frequencies in the rhizosphere over time were duplicated in cycling experiments in the greenhouse that involved successive plantings of wheat in Rhizoctonia-inoculated soil. Chryseobacterium soldanellicola was isolated from the rhizosphere inside the patches and exhibited significant antagonism against R. solani AG-8 in vitro and in greenhouse tests. In conclusion, we identified novel bacterial taxa that respond to conditions affecting bare patch disease symptoms and that may be involved in suppression of Rhizoctonia root rot and bare batch disease.     

Conventional farming impairs Rhizoctonia solani disease suppression by disrupting soil food web

Intensive farming in agriculture raises questions in relation to environmental sustainability and the widespread use of agrochemicals. In the present work, we compare the impact of organic and intensive farming, in connection to the soil suppressiveness against the soilborne pathogen Rhizoctonia solani. Three farms were considered in the study: two practicing organic cultivation (for 10 and 20 years, respectively), and one applying conventional cultivation. Soil suppressiveness was assessed in a greenhouse bioassay with lettuce (Lactuca sativa). Soil microbiome was characterized by combining BIOLOG EcoPlates^? with high‐throughput sequencing of bacterial and eukaryotic rRNA gene markers. Suppressiveness towards R. solani was higher in organic than in conventional farming soil, but this property was lost after soil sterilization. Functional biodiversity was significantly higher in the two organic soils, and this parameter was predictive of the suppressiveness towards R. solani. According to our analyses, the overall microbial taxonomic diversity was unlinked to suppressiveness. A correlation analysis, carried out at the genus level for the most abundant bacterial and eukaryotic microbial taxa, showed that 58.7% of the genera had a statistically significant correlation with suppressiveness. In particular, the genera Flavisolibacter, Massilia, Pseudomonas, Ramlibacter, Rhizophus and the oligochaete worms belonging to the Enchytraeidae family positively correlated with the disease suppression.     

Microbial and biochemical basis of a Fusarium wilt-suppressive soil

Crops lack genetic resistance to most necrotrophic pathogens. To compensate for this disadvantage, plants recruit antagonistic members of the soil microbiome to defend their roots against pathogens and other pests. The best examples of this microbially based defense of roots are observed in disease-suppressive soils in which suppressiveness is induced by continuously growing crops that are susceptible to a pathogen, but the molecular basis of most is poorly understood. Here we report the microbial characterization of a Korean soil with specific suppressiveness to Fusarium wilt of strawberry. In this soil, an attack on strawberry roots by Fusarium oxysporum results in a response by microbial defenders, of which members of the Actinobacteria appear to have a key role. We also identify Streptomyces genes responsible for the ribosomal synthesis of a novel heat-stable antifungal thiopeptide antibiotic inhibitory to F. oxysporum and the antibiotic''s mode of action against fungal cell wall biosynthesis. Both classical- and community-oriented approaches were required to dissect this suppressive soil from the field to the molecular level, and the results highlight the role of natural antibiotics as weapons in the microbial warfare in the rhizosphere that is integral to plant health, vigor and development.     

Biocontrol of Rhizoctonia solani AG-2, the causal agent of damping-off by Muscodor cinnamomi CMU-Cib 461

Rhizoctonia solani is a damping-off pathogen that causes significant crop loss worldwide. In this study, the potential of Muscodor cinnamomi, a new species of endophytic fungus for controlling R. solani AG-2 damping-off disease of plant seedlings by biological fumigation was investigated. In vitro tests showed that M. cinnamomi volatile compounds inhibited mycelial growth of pathogens. Among nine solid media tested, rye grain was the best grain for inoculum production. An in vivo experiment of four seedlings, bird pepper, bush bean, garden pea and tomato were conducted. The results indicated that treatment with 30?g of M. cinnamomi inoculum was the minimum dose that caused complete control of damping-off symptoms of all seedlings after one month of planting. The R. solani-infested soil showed the lowest percentage of seed germination. In addition, M. cinnamomi did not cause any disease symptoms. From the results it is clear that M. cinnamomi is effective in controlling R. solani AG-2 both in vitro and in vivo.     

Formulation of a soil biofungicide for control of damping-off of Chinese cabbage (Brassica chinensis) caused by Rhizoctonia solani

A granulate biofungicide named PBGG was developed by combining Pseudomonas boreopolis with Brassica seed pomace, glycerin and sodium alginate. Application of 1.0% (w/w) of PBGG to the soil infested with Rhizoctonia solani AG-4 significantly reduced the percentage of colonization of cabbage seeds by the pathogen and stimulated proliferations of actinomycetes. Among Streptomyces populations, Streptomyces padanus (strain SS-07) and Streptomyces xantholiticus (strain SS-09) were the two most effective strains with strong antagonistic effects to R. solani. Application of 1% (w/w) PBGG in soil significantly increased population densities of S. padanus and S. xantholiticus. Treating soil with S. padanus (strain SS-07) or S. xantholiticus (strain SS-09) alone or in combination with 1% (w/w) PBGG significantly reduced the percentage of colonization of cabbage seeds by R. solani, compared to untreated controls. S. padanus was the most effective agent in reducing the percentage of seed colonization by the pathogen. Results of greenhouse tests showed that the most effective treatment was the amendment of pathogen-infested soil by S. padanus + 1% (w/w) PBGG which resulted in a disease incidence of 6.5–8.6%, compared to 27.8–31.7% for the treatment of S. xantholiticus + 1% (w/w) PBGG, 36.9–38.6% for the treatment of 1% (w/w) PBGG alone, and 61.8–64.8% for the treatment of control (unamended soil). Results of greenhouse and field tests also showed that soil amendment of 1% (w/w) PBGG alone or 1% (w/w) PBGG mixed with S. padanus (strain SS-07) or S. xantholiticus (strain SS-09) was not harmful to seed germination of Chinese cabbage. The study suggests that amendment of R. solani infested soil with S. padanus + 1% (w/w) PBGG prior to sowing is an effective method for control of damping-off of Chinese cabbage.     

Black scurf of potato

Black scurf of Potato is a serious disease commonly observed in most potato-producing areas of the world. Caused by Rhizoctonia solani AG-3 (teleomorph Thanatephorus cucumeris [Frank] Donk), this disease is favoured by the capacity of fungus to survive in soil as sclerotia and mycelium in plant debris for long periods, and environmental conditions of low soil temperature and high soil moisture. Management of the disease requires an integrated approach since no single tactic is totally effective. An effective control program combines cultural practices, fungicides, biological control, and solarization.     

Identification and evaluation of a potential biocontrol agent,Bacillus subtilis,against Fusarium sp. in apple seedlings

To find a potential biocontrol agent against Fusarium sp. in apple seedlings, an endophytic bacterium strain was isolated from apple tree tissues. The inhibitive efficiency of the isolated strain against the hyphal growth of Fusarium sp. and Rhizoctonia solani was tested. Strain Y-1 showed significant inhibitory effects against Fusarium oxysporum, F. moniliforme, F. proliferatum, F. solani and R. solani. Its antifungal activity against F. oxysporum was the highest, reaching up to 64.90 %. In vivo tests indicated that strain Y-1 effectively protects apple from F. oxysporum infections. The control effect reached 92.26 % when bacterial inoculation was performed 3 days prior to pathogen inoculation. Strain Y-1 could colonize the rhizosphere and tissues within 30 days. It was also able to induce systemic resistance in apple seedlings as shown by the activities of SOD and POD. Strain Y-1 significantly increased the root length, root wet and dry weights, and plant height of the apple seedlings compared with the control group. The homology analysis of the 16S rRNA sequence, together with morphological, physical, and biochemical analyses, revealed that strain Y-1 is Bacillus subtilis.     

Soil Type Dependent Rhizosphere Competence and Biocontrol of Two Bacterial Inoculant Strains and Their Effects on the Rhizosphere Microbial Community of Field-Grown Lettuce

Rhizosphere competence of bacterial inoculants is assumed to be important for successful biocontrol. Knowledge of factors influencing rhizosphere competence under field conditions is largely lacking. The present study is aimed to unravel the effects of soil types on the rhizosphere competence and biocontrol activity of the two inoculant strains Pseudomonas jessenii RU47 and Serratia plymuthica 3Re4-18 in field-grown lettuce in soils inoculated with Rhizoctonia solani AG1-IB or not. Two independent experiments were carried out in 2011 on an experimental plot system with three soil types sharing the same cropping history and weather conditions for more than 10 years. Rifampicin resistant mutants of the inoculants were used to evaluate their colonization in the rhizosphere of lettuce. The rhizosphere bacterial community structure was analyzed by denaturing gradient gel electrophoresis of 16S rRNA gene fragments amplified from total community DNA to get insights into the effects of the inoculants and R. solani on the indigenous rhizosphere bacterial communities. Both inoculants showed a good colonization ability of the rhizosphere of lettuce with more than 10⁶ colony forming units per g root dry mass two weeks after planting. An effect of the soil type on rhizosphere competence was observed for 3Re4-18 but not for RU47. In both experiments a comparable rhizosphere competence was observed and in the presence of the inoculants disease symptoms were either significantly reduced, or at least a non-significant trend was shown. Disease severity was highest in diluvial sand followed by alluvial loam and loess loam suggesting that the soil types differed in their conduciveness for bottom rot disease. Compared to effect of the soil type of the rhizosphere bacterial communities, the effects of the pathogen and the inoculants were less pronounced. The soil types had a surprisingly low influence on rhizosphere competence and biocontrol activity while they significantly affected the bottom rot disease severity.     

Rapeseed rotation, compost and biocontrol amendments reduce soilborne diseases and increase tuber yield in organic and conventional potato production systems

Aims

Integrating multiple soil and disease management practices may improve crop productivity and disease control, but potential interactions and limitations need to be determined.

Methods

Three different potential disease-suppressive management practices, including a Brassica napus (rapeseed) green manure rotation crop, conifer-based compost amendment, and three biological control organisms (Trichoderma virens, Bacillus subtilis, and Rhizoctonia solani hypovirulent isolate Rhs1A1) were evaluated alone and in combination at sites with both organic and conventional management histories for their effects on soilborne diseases and tuber yield.

Results

Rapeseed rotation reduced all observed soilborne diseases (stem canker, black scurf, common scab, and silver scurf) by 10 to 52 % in at least one year at both sites. Compost amendment had variable effects on tuber diseases, but consistently increased yield (by 9 to 15 %) at both sites. Biocontrol effects on disease varied, though Rhs1A1 decreased black scurf at the conventional site and T. virens reduced multiple diseases at the organic site in at least one year. Combining rapeseed rotation with compost amendment both reduced disease and increased yield, whereas biocontrol additions produced only marginal additive effects.

Conclusions

Use of these treatments alone, and in combination, can be effective at reducing disease and increasing yield under both conventional and organic production practices.     

Compost amendments enhance peat suppressiveness to Pythium ultimum,Rhizoctonia solani and Sclerotinia minor

Peat is the most common organic material used for the preparation of potting mix because of its homogeneous and favorable agronomic characteristics. However, this organic material is poorly suppressive against soilborne pathogens and fungicides are routinely used to manage damping-off diseases. In the present study, we investigated the suppressive capability of five compost – peat mixtures towards the plant pathogens Pythium ultimum, Rhizoctonia solani and Sclerotinia minor – Lepidium sativum pathosystems. For all organic media, 18 parameters were measured including enzymatic activities (glucanase, N-acetyl-glucosaminidase, chitobiosidase and hydrolysis of fluorescein diacetate), microbiological (BIOLOG® EcoPlates?, culturable bacteria and fungi), and chemical features (pH, EC, total, extractable and humic carbon, total and organic N, NH₄–N, total protein and water content). In addition, ¹³C-CPMAS-NMR spectroscopy was used to characterize the organic materials. Peat amended with composts reduced disease damping-off caused by P. ultimum, R. solani and S. minor in 60% of the mixtures and compost derived from animal manure showed the largest and most consistent disease suppression. Sterilization decreased or eliminated suppressiveness of 42.8% of the mixtures. The most useful parameters to predict disease suppression were different for each pathogen: extractable carbon, O-aryl C and C/N ratio for P. ultimum, alkyl/O-alkyl ratio, N-acetyl-glucosaminidase and chitobiosidase enzymatic activities for R. solani and EC for S. minor. Our results demonstrate that the addition of composts to peat could be useful for the control of soilborne pathogens.     

Selection of biological control agents against tomato Fusarium wilt and evaluation in greenhouse conditions of two selected agents in three growing media

Two biological control practices are the use of suppressive growing media and the application of biological control agents (BCAs). The goals of this study were: (i) to screen 584 potential BCAs obtained from Fusarium wilt (FW) suppressive growing media; (ii) to evaluate in greenhouse conditions selected BCAs in three growing media with different degrees of suppressiveness of tomato FW. Two isolates selected after screening were identified as Fusarium solani (305) and Streptomyces sp. (A19). Results showed that tomato FW was reduced and total production was improved when both BCAs were applied to a conducive medium (coir fiber). In highly suppressive growing medium (grape marc compost), A19 and 305 inoculations did not improve suppressiveness. In moderately suppressive growing medium (cork compost), only A19 improved this compost to natural grape marc compost suppressiveness level. Therefore, compost suppressiveness of tomato FW depended on the nature of the compost and on the isolates applied.     

Identification and pathogenicity of Rhizoctonia species isolated from bean and soybean plants in Samsun,Turkey

A total of 434 isolates of Rhizoctonia belonging to 10 anastomosis groups were obtained from the roots and rhizosphere soils of bean and soybean plants grown in Samsun, Turkey. AG-4 was found to be the most common group on bean and soybean plants and AG-5, AG-6, binucleate AG-A, AG-B and R. zeae were other groups isolated from the both plant species. AG-1, AG-7 and AG-K from bean and AG-E from soybean were other groups obtained in the study. The pathogenicity tests on bean and soybean seedlings showed that the highest disease severities were caused by AG-4 isolates, whereas AG-1 and AG-6 isolates were moderately pathogenic. Binucleate Rhizoctonia AG-B isolates were also moderately pathogenic, while other binucleate Rhizoctonia were found to be weakly pathogenic. Rhizoctonia zeae isolates caused moderate disease symptoms on bean, but soybean plants were slightly affected by this group of isolates. This is the first reported observation of R. solani AG-6 and AG-7 and binucleate Rhizoctonia AG-B on bean, and R. solani AG-5 and AG-6 and binucleate Rhizoctonia AG-A, AG-B and AG-E on soybean, in Turkey.     

A procedure for the metagenomics exploration of disease-suppressive soils

The microbiota of, in particular, disease-suppressive soils contains a wealth of antibiotic biosynthetic loci that are inaccessible by traditional cultivation-based techniques. Hence, we developed a methodology based on soil microbial DNA, which allowed the metagenomics-based unlocking of the relevant genes. Here, a streamlined soil metagenomics protocol is presented. The protocol consists of an optimized method to extract bacterial cells from a Rhizoctonia solani AG3 suppressive loamy sand soil followed by DNA extraction and purification, and the preparation of a clone library in an efficient host/vector system. Methods for the functional and genetic screening of the library for antibiotic production loci are also described. Using the suppressive soil, we thus produced, screened and tested an approximate 15,000-membered metagenomic library of fosmids in an Escherichia coli host. Functional screens, based on dual culturing of clone arrays with R. solani AG3 and Bacillus subtilis 168, were largely negative. Genetic screens, based on hybridizations with soil-generated probes for polyketide biosynthesis, non-ribosomal protein synthesis and gacA, revealed several inserts, of around 40-kb in size, with potential antibiotic production capacity. We present the full sequences of three selected clones. We further examine the challenges that still impinge on the metagenomic exploration of disease-suppressive soil.     

Clonostachys rosea,a potential biological control agent for Rhizoctonia solani AG-3 causing black scurf on potato

Rhizoctonia solani AG-3 forms sclerotia on potato tubers, reducing quality and marketability. Potato discs with sclerotia were exposed to saturated soil, and following toothpick baiting, hyphae of R. solani parasitised with mycelia that either coiled around or penetrated cell walls were observed. Similar parasitic behaviour was observed in dual cultures. Clonostachys rosea was identified as the mycoparasite by morphology and ITS sequencing. When co-inoculated onto stems grown from disease-free seed potatoes, tubers of the C. rosea?+?R. solani inoculated plants had significantly lower black scurf (P?0.0001) and higher yield (P?=?0.0002) than R. solani inoculated controls.     

Biocontrol activity of salt tolerant Streptomyces isolates against phytopathogens causing root rot of sugar beet

Biological control of fungi causing root rot on sugar beet by native Streptomyces isolates (C and S2) was evaluated in this study. The dry weight and colony forming unit (CFU) of S2 and C increased when 300 mM NaCl was added to medium. The in vitro antagonism assays showed that both isolates had inhibitory effect against Rhizoctonia solani AG-2, Fusarium solani and Phytophthora drechsleri. In dual culture, Streptomyces isolate C inhibited mycelial growth of R. solani, F. solani and P. drechsleri 45%, 53% and 26%, respectively. NaCl treatment of medium increased biocontrol activity of soluble and volatile compounds of isolate C and S2. After salt treatment, growth inhibition of R. solani, F. solani and P. drechsleri by isolate C increased up to 59%, 70% and 79%, respectively. To elucidate the mode of antagonism, protease, chitinase, beta glucanase, cellulase, lipase and α-amylase activity and siderophore and salicylic acid (SA) production were evaluated. Both isolates showed protease, chitinase and α-amylase activity. Also, biosynthesis of siderophore was detectable for both isolates. Production of siderophore and activity of protease and α-amylase increased after adding salt for both isolates. In contrast, chitinase activity decreased significantly. Production of SA, beta glucanase and lipase by isolate S2 and biosynthesis of cellulase by isolate C were observed in presence and absence of NaCl. Soil treatment with Streptomyces isolate C inhibited root rot of sugar beet caused by P. drechsleri, R. solani and F. solani. Results of this study showed that these two Streptomyces isolates had potential to be utilized as biocontrol agent against fungal diseases especially in saline soils.     

Molecular and phenotypic characterization of potential plant growth-promoting <Emphasis Type="Italic">Pseudomonas</Emphasis> from rice and maize rhizospheres

In this study, Pseudomonas species were isolated from the rhizospheres of two plant hosts: rice (Oryza sativa cultivar Pathum Thani 1) and maize (Zea mays cultivar DK888). The genotypic diversity of isolates was determined on basis of amplified rDNA restriction analysis (ARDRA). This analysis showed that both plant varieties selected for two distinct populations of Pseudomonas. The actual biocontrol and plant promotion abilities of these strains was confirmed by bioassays on fungal (Verticillum sp., Rhizoctonia solani and Fusarium sp.) and bacterial (Ralstonia solanacearum and Bacillus subtilis) plant pathogens, as well as indole-3-acetic acid (IAA) production and carbon source utilization. There was a significant difference between isolates from rice and maize rhizosphere in terms of biological control against R.  solanacearum and B.  subtilis. Interestingly, none of the pseudomonads isolated from maize rhizosphere showed antagonistic activity against R.  solanacearum. This study indicated that the percentage of pseudomonad isolates obtained from rice rhizosphere which showed the ability to produce fluorescent pigments was almost threefold higher than pseudomonad isolates obtained from maize rhizosphere. Furthermore, the biocontrol assay results indicated that pseudomonad isolated from rice showed a higher ability to control bacterial and fungal root pathogens than pseudomonad isolates obtained from maize. This work clearly identified a number of isolates with potential for use as plant growth-promoting and biocontrol agents on rice and maize.