Rhizoctonia Solani AG Research Articles

Effect of Brassicaceae seed meals with different glucosinolate profiles on Rhizoctonia root rot in wheat

The soil-borne pathogen Rhizoctonia solani AG 8 causes major yield losses in wheat (Triticum aestivum. L) production worldwide. Plant tissues of Brassicaceae species contain glucosinolates that are hydrolyzed in the presence of the enzyme myrosinase into products with pesticidal properties. Growth chamber studies were conducted to determine the effect of the Brassicaceae seed meals (SMs) from Brassica juncea, Brassica napus and Sinapis alba on the suppression of the R. solani AG 8 infection of winter wheat. Pasteurized sandy soils were amended with intact and denatured SMs of rape seed and mustard at a rate of 0.5% by soil weight. Regardless of the glucosinolate type and content, all intact and denatured Brassicaceae significantly reduced the infection of winter wheat seedlings by R. solani AG 8 compared to the un-amended control. However, soils amended with S. alba SMs had the lowest severity of Rhizoctonia root rot relative to other amended soils. Phytotoxicity arising from the use of Brassicaceae SMs was observed particularly in soils amended with high glucosinolate-containing SMs. These studies demonstrate that Brassicaceae SMs can be used to manage disease caused by R. solani AG-8. However, future studies will need to focus on strategies for diminishing the crop growth-reducing effects associated with Brassicaceae SM amendment to fully maximize these fungicidal benefits.

Mechanism of the Generation of New Somatic Compatibility Groups within <i>Thanatephorus cucumeris</i> (<i>Rhizoctonia solani</i>)

Single-basidiospore isolates (SBIs) were obtained from field isolates of Thanatephorus cucumeris (Rhizoctonia solani) AG-1 IC and AG-2-2 IV. Formation of distinctive tufts, a recognized feature of heterokaryon synthesis, was observed, and isolates derived from hyphal-tipped tuft hyphae were obtained following pairings between various strains. Three distinctive types of tufts were formed: the fibrous type of mating-compatible homokaryon-homokaryon (Hom-Hom) pairings, the sparse type between heterokaryon-homokaryon (Het-Hom) pairings originating from one parent, and the compact type between Het-Hom pairings originating from different parents. Amplified Fragment Length Polymorphism (AFLP) profile of fingerprints of these tuft isolates verified that they were all heterokaryotic. Because of heterokaryotic vigor, the growth and pathogenicity of the majority of tuft isolates increased compared with their contributing SBIs. New somatic compatibility groups (SCGs) that were different from parental field isolates occurred following heterokaryon formation within T. cucumeris. Tuft isolates produced by Hom-Hom and Het-Hom pairings among isolates of different parents yielded no somatic compatibility with the original parent isolates and a high frequency of new SCGs (62–100%). This was in contrast to those produced by Hom-Hom and Het-Hom pairings among isolates with a common parent that yielded only 12–37% new SCGs. The SCG diversity of R. solani in the field may be attributed to new fitter heterokaryons formed between a heterokaryon of one pair of parents and a homokaryon of another parent pair. This mechanism greatly contributes to genetic diversity in the field and accounts for the failure to recover the expected distribution of SCGs from a field population.

Rhizoctonia solani AG 2-2IIIB에 의한 마 뿌리썩음병의 한국 내 발생

2011년 안동과 진주의 마 재배포장에서 줄기 지제부 및 괴근썩음 증상이 나타났다. 병징을 나타내는 부위로부터 Rhizoctonia와 유사 속에 속하는 20개 균주가 분리되었다. rDNA-internal transcribed spacer(ITS) 염기서열 상동성을 기초로 8균주는 Rhizoctonia solani, 12균주는 Ceratobasidium sp.로 동정되었다. rDNA-ITS 염기서열의 cluster 분석에 의해 R. solani에 속하는 8개 균주 중 7개 균주는 균사융합군 AG 2-2IIIB, 1균주는 AG 1-1A에 속하였다. 또한, Ceratobasidium sp.에 속하는 12균주 중 7균주는 AG-Fa, 3균주는 AG-A, 나머지 2균주는 각각 AG-Fb와 AG-O에 속하였다. R. solani AG 2-2IIIB 균주들은 마의 줄기와 괴근에 병원성이었으나 R. solani AG 1-1A와 모든 Ceratobasidium sp. 균주는 비병원성이라는 것이 확인되었다. 이 결과는 조사지역에서 R. solani AG 2-2IIIB가 마의 줄기 및 괴근썩음병을 일으키는 중요한 병원균이라는 것을 나타낸다. 이 연구는 국내에서 R. solani AG 2-2IIIB에 의한 마 뿌리썩음병에 대하여 처음으로 보고하는 것이다. Stem canker and tuber rot symptoms were observed on yam grown in Andong and Jinju, Korea in 2011. A total of 20 isolates of Rhizoctonia and allied fungi were obtained from the symptomatic plants. Among the isolates, 8 isolates were identified as Rhizoctonia solani and 12 isolates as Ceratobasidium sp. based on rDNA-internal transcribed spacer (ITS) sequence similarity. In the cluster analysis of rDNA-ITS sequences, 7 isolates of R. solani belonged to AG 2-2IIIB and remaining one to AG 1-1A. In addition, among the 12 isolates of Ceratobasidium sp., 7 isolates belonged to AG-Fa, three isolates to AG-A and the other two isolates to AG-Fb and AG-O, respectively. Pathogenicity tests showed that all the R. solani AG 2-2IIIB isolates are pathogenic on stem and tuber of yam but R. solani AG 1-1A and all the Ceratobasidium isolates are non-pathogenic. The results indicate that R. solani AG 2-2IIIB is an important pathogen causing stem canker and tuber rot on yams grown in the study areas. This is the first report of R. solani AG 2-2IIIB causing stem canker and tuber rot of yam in Korea.

ANTIOXIDANT SYSTEMS IN SOYBEAN AND MAIZE SEEDLINGS INFECTED WITH RHIZOCTONIA SOLANI

SUMMARY Changes in enzymatic and non-enzymatic antioxidant systems in soybean (cv. Becejka) and maize (hybrid NS 640) seedlings were studied after inoculation with Rhizoctonia solani AG 2-2 IIIB. Enzymes such as plant peroxidases and phenylalanine ammonia-lyase were induced in the analyzed organs of both plant species. Nevertheless, increase in superoxide dismutase activity was recorded only in infected maize roots. After inoculation, lipid peroxidation was enhanced only in soybean plants. Seedlings showed ability to accumulate phenolic compounds, some of which correlated with disease intensity. A significant decrease in total carotenoid amounts was recorded in infected soybean and maize plants. After R. solani infection, the enzymatic and nonenzymatic antioxidant systems were more efficient in maize than in soybean seedlings.

A new large scale soil DNA extraction procedure and real-time PCR assay for the detection of Sclerotium cepivorum in soil

A real-time PCR assay specific for Sclerotium cepivorum, the causal agent of white rot in onions, was developed for use with a new DNA extraction method capable of processing up to 1 kg of soil in weight. The assay was specific for S. cepivorum when tested against 24 isolates representative of 14 closely related species and other pathogens of onion. The assay was highly sensitive when used with soil DNA extracted using the new DNA extraction procedure. In three different field soils tested, a good relationship between cycle threshold (Ct) and number of sclerotia was observed (R² = 0.89). Twenty-nine soil samples from onion and leek crops were obtained and the pathogen was detected in four samples. All four positive samples were associated with current or past outbreaks of white rot of onion. Additional assays were also used on the 29 field soil samples, Botrytis aclada and Rhizoctonia solani AG8 were also detected, in one soil sample each. Rhizoctonia solani AG2-1 was more widespread and was detected in eight different soil samples. The method is therefore suitable to quantify levels of S. cepivorum in soil samples, with the added advantage that the technique allows other soil pathogens of interest to be assayed from the same DNA sample. The bulk soil DNA extraction method described here has the potential to be used to detect soil-borne pests and pathogens for other crops in a wide range of soil types.

Gene expression profiling of the plant pathogenic basidiomycetous fungus Rhizoctonia solani AG 4 reveals putative virulence factors

Rhizoctonia solani is a ubiquitous basidiomycetous soilborne fungal pathogen causing damping-off of seedlings, aerial blights and postharvest diseases. To gain insight into the molecular mechanisms of pathogenesis a global approach based on analysis of expressed sequence tags (ESTs) was undertaken. To get broad gene-expression coverage, two normalized EST libraries were developed from mycelia grown under high nitrogen-induced virulent and low nitrogen/methylglucose-induced hypovirulent conditions. A pilot-scale assessment of gene diversity was made from the sequence analyses of the two libraries. A total of 2280 cDNA clones was sequenced that corresponded to 220 unique sequence sets or clusters (contigs) and 805 singlets, making up a total of 1025 unique genes identified from the two virulence-differentiated cDNA libraries. From the total sequences, 295 genes (38.7%) exhibited strong similarities with genes in public databases and were categorized into 11 functional groups. Approximately 61.3% of the R. solani ESTs have no apparent homologs in publicly available fungal genome databases and are considered unique genes. We have identified several cDNAs with potential roles in fungal pathogenicity, virulence, signal transduction, vegetative incompatibility and mating, drug resistance, lignin degradation, bioremediation and morphological differentiation. A codon-usage table has been formulated based on 14694 R. solani EST codons. Further analysis of ESTs might provide insights into virulence mechanisms of R. solani AG 4 as well as roles of these genes in development, saprophytic colonization and ecological adaptation of this important fungal plant pathogen.

Elucidation of Antifungal Metabolites Produced by Pseudomonas aurantiaca IB5-10 with Broad-Spectrum Antifungal Activity

Antifungal metabolites were isolated from a culture of Pseudomonas aurantiaca IB5-10. Chemical structures of the metabolites were elucidated as phenazine-1-carboxylic acid (PCA; 1), 2-hydroxyphenazine (2-OH-PHZ; 2), and cyclo-(L-Pro-L-Val; 3), respectively, based on spectroscopic methods. Among them, 3 was isolated for the first time from this strain. The antifungal activities of 1-3 were evaluated against a variety of plant pathogens. To the best of our knowledge, the antifungal activities of 3 against plant fungal pathogens have been evaluated for the first time in this work. PCA (1) showed the most potent antifungal activities against Phytophthora capsici, Rhizoctonia solani AG-1(IA), and Pythium ultimum with MICs (microgram/ml) of less than 1.0, 1.3, and 2.0, respectively. On the other hand, 2-OH-PHZ (2) showed potent antifungal activity against R. solani AG-1(IA) with the MIC (microgram/ml) of 2.0, whereas it showed moderate antifungal activity against P. ultimum with the MIC (microgram/ml) of 50.0. In addition, 3 showed antifungal activity against only R. solani AG- 1(IA).

Interactions between the root pathogen Rhizoctonia solani AG‐8 and acetolactate‐synthase‐inhibiting herbicides in barley

The widespread acceptance of reduced-tillage farming in cereal cropping systems in the Pacific Northwest of the United States has resulted in increased use of herbicides for weed control. However, soil residual concentrations of widely used imidazalone herbicides limit the cultivation of barley, which is more sensitive than wheat. In addition, increased severity of the root rot disease caused by Rhizoctonia solani is associated with reduction in tillage. Many crops exhibit altered disease responses after application of registered herbicides. In this study, the injury symptoms in barley caused by sublethal rates of two acetolactate synthase (ALS)-inhibiting herbicides, imazamox and propoxycarbazone-sodium, were assessed in factorial combinations with a range of inoculum concentrations of the root rot pathogen Rhizoctonia solani AG-8. Both herbicides and pathogen had negative impacts on plant growth parameters such as root and shoot dry weight, shoot height and first leaf length, and interactions between pathogen and herbicide were detected. The results suggested that sublethal rates of herbicides and R. solani could alter severity of injury symptoms, possibly owing to the herbicide predisposing the plant to the pathogen.

Establishment of Agrobacterium tumefaciens- Mediated Transformation System for Rice Sheath Blight Pathogen Rhizoctonia solani

To construct the T-DNA insertional mutagenesis transformation system for rice sheath blight pathogen Rhizoctonia solani AG-1 IA, the virulent isolate GD118 of this pathogen was selected as an initial isolate for transformation. The conditions for transformation of isolate GD118 were optimized in five aspects, i.e. pre-induction time, co-culture time, acetosyringone (AS) concentration at the co-culture phase, co-culture temperature and pH value of induction solid medium (ISM) at the co-culture phase. Finally, a system of Agrobacterium tumefaciens-mediated transformation (ATMT) for R. solani AG-1 IA was established successfully. The optimal conditions for this ATMT system were as follows: the concentration of hygromycin B at 30 μg/mL for transformant screening, 8 h of pre-induction, 20 h of co-culture, 200 μmol/L of AS in ISM, co-culture at 25 °C and pH 5.6 to 5.8 of ISM at the co-culture phase. The transformants still displayed high resistance to hygromycin B after subculture for five generations. A total of 10 randomly selected transformants were used for PCR verification using the specific primers designed for the hph gene, and the results revealed that an expected band of 500 bp was amplified from all of the 10 transformants. Moreover, PCR amplification for these 10 transformants was carried out using specific primers designed for the Vir gene of A. tumefaciens, with four strains of A. tumefaciens as positive controls for eliminating the false-positive caused by the contamination of A. tumefaciens. An expected band of 730 bp was amplified from the four strains of A. tumefaciens, whereas no corresponding DNA band could be amplified from the 10 transformants. The results of the two PCR amplifications clearly showed that T-DNA was indeed inserted into the genome of target isolate GD118.

First Report of Rhizoctonia solani AG 8 on Wheat in Turkey

A survey was conducted in Ankara and Eskisehir provinces of Turkey for determining anastomosis groups and pathogenicity of Rhizoctonia species associated with root and crown rot of wheat. Pathogenicity tests revealed that Rhizoctonia solani AG 8 caused the common symptoms of damping-off and stunting.

Efeitos da simulação da solarização do solo com materiais vegetais sobre o crescimento micelial de fungos fitopatogênicos habitantes do solo

A incorporação de materiais vegetais específicos associados à solarização do solo tem sido um avanço promissor no controle de fungos fitopatogênicos habitantes do solo. O objetivo do trabalho foi avaliar determinados efeitos da incorporação e decomposição de brócolis, mamona, mandioca brava e mansa, no solo, em condições de microcosmo mantido em BOD (37±2ºC), sobre o micélio de Fusarium oxysporum f. sp. lycopersici Raça 2, Macrophomina phaseolina, Rhizoctonia solani AG-4 HGI e de Sclerotium rolfsii. Assim, quatro ensaios idênticos foram instalados em conjunto de microcosmos, com cinco tratamentos e quatro períodos de tempo diferentes e independentes (7, 14, 21 e 28 dias). O parâmetro avaliado foi os efeitos inócuo, fungistático e fungicida dos tratamentos sobre o micélio dos fungos. Verificou-se efeito fungistático e fungicida no crescimento micelial de F. oxysporum f. sp. lycopersici Raça 2, R. solani AG-4 HGI e de S. rolfsii. Os fungos que apresentaram efeito fungistático apresentaram uma velocidade média de crescimento micelial inferior ao controle geral, que consistiu na incubação dos fungos em temperatura de 25±2ºC. O efeito fungicida ocorreu aos 21 dias de incubação para F. oxysporum e R. solani e aos 28 dias para S. rolfsii. Para M. phaseolina, observou-se apenas efeito inócuo. Associação da temperatura de 37±2ºC mais o período de tempo dos tratamentos foi o fator responsável pelos efeitos fungistático e fungicida no micélio dos fitopatógenos estudados. Essa associação também interferiu na velocidade do crescimento micelial dos fungos que apresentaram efeito fungistático.

English

The effect of increased concentrations of potassium bicarbonate (KHCO3) as a possible alternative to synthetic fungicides for controlling Rhizoctonia solani AG 4 HG-I and Sclerotinia sclerotiorum was evaluated in vitro, in this study. In addition, the effect of potassium bicarbonate on Trichoderma sp., a natural antagonist on R. solani AG 4 HG-I and S. sclerotiorum was determined. Potassium bicarbonate substantially inhibited (P<0.05) the growth of the three fungal strains. Mycelial growth of R. solani AG 4 HG-I significantly decreased as the concentration of bicarbonate increased, especially at concentrations greater than 200 mM. Similarly, mycelial growth of both S. sclerotiorum and Trichoderma sp. dramatically reduced in increasing concentrations of KHCO3. Mycelial growth of either fungi was completely inhibited when exposed to 100 mM bicarbonate. In addition, KHCO3concentrations higher than 10 mM caused significant (P<0.05) reduction of the sclerotium formation of S. sclerotiorum. Also, sclerotium germination and de novosclerotium formation were significantly inhibited as the concentrations of KHCO3increased. As a result, it was concluded that potassium bicarbonate was an alternative chemical agent for controlling R. solani AG 4 HG-I and S. sclerotiorum.Also, KHCO3 was found to have negative effects on Trichoderma sp.   Key words: Antifungal effect, KHCO3, soil borne pathogens, sclerotium germination.

The plant growth-promoting fungus Fusarium equiseti and the arbuscular mycorrhizal fungus Glomus mosseae stimulate plant growth and reduce severity of anthracnose and damping-off diseases in cucumber (Cucumis sativus) seedlings

To alleviate the environmental contamination due to persistent chemical usage, approaches to integrated pest management were conceived. In this perspective, microbe–microbe interactions such as mycorrhizal relationships with other soil microbiota in the rhizosphere like the plant growth-promoting fungi (PGPF) are particularly important. Better understanding of the interactions between beneficial microbial groups is imperative in the identification of possible synergistic or antagonistic effects to improve their practical usage as biocontrol agents or biofertilizers. In this study, the consequence of co-inoculation of the arbuscular mycorrhizal fungus (AMF) Glomus mosseae (Gm) and the PGPF Fusarium equiseti (isolates GF18-3 and GF19-1) in terms of plant growth enhancement, root and rhizosphere colonisation, and development of anthracnose (Colletotrichum orbiculare) and damping-off (Rhizoctonia solani AG-4) diseases in cucumber plants was investigated under controlled conditions. The amendment of either GF18-3 or GF19-1 singly or in combination with Gm indicated a general tendency to significantly enhance the shoot dry weight (SDW) of cucumber plants at 4 weeks after planting (WAP). Similarly, Gm alone significantly enhanced SDW at 4 WAP. Gm showed a tendency to depress root colonisation by F. equiseti but such antagonistic effect was not observed in the rhizosphere soil. Both GF18-3 and GF19-1 significantly reduced percent root colonisation of Gm. However, these general tendencies may vary with the inoculum densities of AMF and PGPF. Both F. equiseti and Gm inoculated singly significantly increased percent of protection against anthracnose, but the combined inoculation was more effective in controlling the disease compared to single inoculation. The inoculation of the cucumber seedlings with GF18-3, GF19-1 or Gm, 6 or 12 days prior to damping-off pathogen inoculation, increased percent of protection against damping-off disease. This study shows that the co-inoculation of F. equiseti and Gm resulted in additive effect on the suppression of anthracnose disease in cucumber.

밀 잎집눈무늬병원균(Rhizoctonia cerealis)의 배양적 특성과 국내육성 맥류 품종의 저항성 평가

밀의 잎의 황화와 줄기 고사 증상이 발생한 포장에서 분리한 균을 동정한 결과 배지 생육 반응, 형태학적 유전자적 특성과 병원성, 균의 DNA 검정 결과 밀 잎집눈무늬병을 일으키는 Rhizoctonia cerealis로 확인되었다. R. cerealis의 온도와 pH에 따른 배양적 특성을 조사한 결과 생육 최적 온도는 <TEX>$20{\sim}25^{\circ}C$</TEX>로 약간 저온성인 것으로 확인 되었다. pH에 따른 생육 반응 조사 결과는 pH 5~7에서 생육이 좋아 대체로 약산성에서 활발한 특징을 보였다. 밀 잎집눈무늬병에 대해 국내 육성 맥류 품종에 대한 저항성 검정을 수행하였다. 밀 품종 검정 결과 검정한 29품종에서 20% 이상의 발병률로 저항성이 확인되지 않았다. 안백밀 등 12품종이 발병률 20~30%의 중도저항성을 보였으나 '그루밀' 등 17품종은 40~80% 이상으로 감수성을 보였다. 사료용으로 이용되는 청보리, 귀리, 호밀, 트리티케일 검정 결과에서는 호밀 2품종에서만 50% 이상의 발병률을 모여 감수성으로 나타났으나, 청보리, 귀리, 트리티케일에서는 발병율 15% 이하로 저항성인 것으로 나타났다. 귀리나 트리티케일, 호밀의 발병과 저항성 평가는 국내에서 처음 이루어졌다. 한편 식용 및 맥주용 보리 검정 결과 겉보리 9품종 중 '탑골보리', '알보리', '서둔찰보리' 등 3품종이 발병률 10% 이하의 저항성을 보였으며 6개 겉보리와 5개 맥주보리 전체 검정 품종은 중도 저항성을 보인 반면 검정 쌀보리 품종들은 감수성인 것으로 나타났다. It was identified as a sharp eyespot (Rhizoctonia cerealis) that the isolates from abnormal symptoms in wheat that showed yellowing leaves, necrotic spot on stem base and dead tillers. These isolates have slower growth property and fewer mycelia than Rhizoctonia solani AG-1(1A) (KACC 40106). They showed binuclear cell, same media cultural and DNA characteristics to R. cerealis. They caused same symptoms on leaves and stem base appeared in artificial inoculation test, comparing to diseased wheat fields and also affect to maturing of kernels. They have optimal growth temperature and acidity on the artificial media as <TEX>$20{\sim}25^{\circ}C$</TEX> and pH 5~7, respectively. In the investigation of varietal resistance of Korean winter cereal crops to sharp eyespot, there was no resistant in wheat cultivars that all materials infected over 20% diseased ratio. 12 cultivars including 'Anbaekmil', however, considered to moderate resistance with 20 to 30% infection ratio. The others crops using in feeding, whole crop barley, oat, rye and triticale were resistant below 15% diseased degree except the rye that showed over 50% infection rate. It was the first evaluation to sharp eyespot resistance for the Korean feeding crop cultivars. Most tested Korean barley cultivars for malting and food were moderate and susceptible to the sharp eyespot. Only 3 hulled barley, 'Tapgolbori', 'Albori' and 'Seodunchalbori', showed resistance with less than 10% diseased ratio. All tested naked barley cultivars showed susceptible response to the disease.

Effects of Pseudomonas aureofaciens 63-28 on Defense Responses in Soybean Plants Infected by Rhizoctonia solani

The objective of this work was to investigate the ability of the plant growth-promoting rhizobacterium Pseudomonas aureofaciens 63-28 to induce plant defense systems, including defense-related enzyme levels and expression of defense-related isoenzymes, and isoflavone production, leading to improved resistance to the phytopathogen Rhizoctonia solani AG-4 in soybean seedlings. Seven-dayold soybean seedlings were inoculated with P. aureofaciens 63-28, R. solani AG-4, or P. aureofaciens 63-28 plus R. solani AG-4 (P+R), or not inoculated (control). After 7 days of incubation, roots treated with R. solani AG-4 had obvious damping-off symptoms, but P+R-treated soybean plants had less disease development, indicating suppression of R. solani AG-4 in soybean seedlings. Superoxide dismutase (SOD) and catalase (CAT) activities of R. solani AG-4-treated roots increased by 24.6% and 54.0%, respectively, compared with control roots. Ascorbate peroxidase (APX) and phenylalanine ammonia lyase (PAL) activities of R. solani AG-4-treated roots were increased by 75.1% and 23.6%, respectively. Polyphenol oxidase (PPO) activity in soybean roots challenged with P. aureofaciens 63-28 and P+R increased by 25.0% and 11.6%, respectively. Mn-SOD (S1 band on gel) and Fe-SOD (S2) were strongly induced in P+R-treated roots, whereas one CAT (C1) and one APX (A3) were strongly induced in R. solani AG-4- treated roots. The total isoflavone concentration in P+Rtreated shoots was 27.2% greater than the control treatment. The isoflavone yield of R. solani AG-4-treated shoots was 60.9% less than the control.

Evaluation of the Biocontrol Potential of Some Medicinal Plant Materials Alone and in Combination with Trichoderma harzianum Against Rhizoctonia solani AG 2-1

Fifty five species of medicinal plant materials were tested for their antifungal activity in vitro against Rhizoctonia solani AG 2-1 and Trichoderma harzianum to select plant species that can be used to improve the biocontrol efficacy of T. harzianum. Six species were effective against R. solani AG 2-1 but were also antagonistic to T. harzianum, except for Cinnamomum loureirii stem bark (CSB). CSB inhibited mycelial growth of R. solani AG 2-1 by 73.7% but showed an inhibitory effect on mycelial growth of T. harzianum by only 2.2%. Scanning electron microscophs showed that the CSB treatment resulted in deformed R. solani AG 2-1 hyphal cells, and transmission electron microscophs revealed degenerated cell structures such as degenerated cytoplasm and disentangled cell wall and the accumulation of electron-dense inclusions (asterisks) in the CSB treatment. The biocontrol efficacy of radish damping-off increased greatly following the combined treatments of T. harzianum and CSB and the combined treatment increased efficacy from 6.4-23.1% to 37.1-87.3% compared with either treatment alone. CSB did not affect T. harzianum population growth, as it was almost the same in rice-bran peat medium (culture) amended with 0.1% and 1.0% CSB powder as in non-amended medium. The formulation of T. harzianum in rice-bran peat medium amended with CSB powder reduced the severity of radish damping-off by 80.6%, suggesting that T. harzianum and CSB can be formulated as a biocontrol product for the control of R. solani AG 2-1.

Evaluation of biocontrol agro-techniques against R. solani: study of microbial communities catabolic profile modifications

SUMMARYDamping off is the most common disease caused by edaphic fungi in Spanish crops, among which Rhizoctonia solani AG-4 stands out. In the present work, two possible methods of control were evaluated, incorporation of different doses of organic matter (OM; obtained from strawberry crops) and Pseudomonas fluorescens as a plant growth promoting rhizobacteria (PGPR). The highest inhibition (43% less) against the pathogen was found in the assays that used 20 g of biofumigant/kg soil. Inoculation of the P. fluorescens strain (PGPR) did not protect against the pathogen. In addition, the microbial evolution during incubation with OM was studied. For this purpose, the bacterial and fungal catabolic profiles were determined (using Biolog Eco and FF plates, respectively) as well as bacterial counts of total aerobes, Pseudomonas sp. and aminocyclopropane-1-carboxylate (ACC)-degrading populations, during OM incorporation. This agro-technique produced changes in microbial catabolic community profiles, increasing bacterial metabolic activity and minimizing metabolic diversity of micro-organisms under control with and without pathogen. As for microbial counts, aerobic and ACC-degrading populations decreased while Pseudomonas sp. population increased with OM treatments.The OM amendment applied to control the damping off caused by R. solani is viable; it is more environmentally friendly and has a lower economic cost than chemical controls and, therefore, it could serve as a component in integrated-management programmes.

Impact of Continuous Cropping of Lettuce on the Disease Dynamics of Bottom Rot and Genotypic Diversity of Rhizoctonia solani AG 1-IB

The impact of continuous cropping of lettuce on the disease dynamics of bottom rot and genotypic diversity of the causal pathogen Rhizoctonia solani AG 1-IB was studied over 3 years with two crops per year within a field naturally infested with R. solani the pathogen. This field had not had lettuce cultivated in it for 7 years. The disease incidence (DI) and disease severity (DS) were assessed at each harvest and mapped. Surprisingly, a high DI was already observed in the first crop of year one of this field study. In addition, the pathogen was also found to be evenly distributed. Severely infected plants occurred mainly in patches, and the position varied between crops. A significant increase in DS was already observed in the second year, and both temperature conditions and continuous cropping influenced the DS on average over time. Rhizoctonia isolates were randomly collected from the first crop in 1999 and the sixth crop in 2001. The genotypic diversity within the subgroup of R. solani AG 1-IB was analysed by BOX-PCR genomic fingerprinting and the aggressiveness of isolates by bioassay. The fingerprints revealed a high level of genotypic diversity within the AG 1-IB field population. However, continuous cropping was found not to have an impact on genotypic diversity and aggressiveness.

Characterization of Rhizoctonia spp. Isolates Associated with Damping-Off Disease in Cotton and Tobacco Seedlings in Greece.

Isolates of Rhizoctonia spp. were obtained during the spring of 2007 from diseased cotton and tobacco seedlings showing damping-off symptoms. The sampled fields were located in the main cotton- and tobacco-cultivating regions of central and northern Greece. Among the 79 isolates obtained from cotton plants, 17 were binucleate and 62 were multinucleate whereas, among the 89 isolates obtained from tobacco plants, 87 were multinucleate and only 2 were binucleate. Characterization of anastomosis groups (AGs) was performed with hyphal anastomosis reactions using tester isolates of known AG groups. Anastomosis reactions in cotton mutlinucleate isolates showed that 54 of them belonged in Rhizoctonia solani AG-4, 6 in AG-7, 1 in AG-2, and 1 in AG-3. In the 87 tobacco multinucleate isolates, anastomosis reactions showed that 70 of them belonged in R. solani AG-2, 16 in AG-4, and 1 in AG-5. In addition, molecular characterization was carried out using the specific ribosomal DNA internal transcribed spacer region, in a randomly selected number of isolates. In cotton, the most prevalent AG was AG-4, with 18 isolates to the subgroup HG-I, 1 isolate to the subgroup HG-II, and 7 isolates to the subgroup HG-III, followed by AG-7 (7 isolates), AG-2-1 (1 isolate), and AG-3 (1 isolate). In tobacco, the most prevalent group was AG-2-1 (70 isolates), followed by AG-4 (6 isolates to the subgroup HG-I and 5 isolates to the subgroup HG-III) and a single isolate belonging to AG-5. Phylogenetic analysis showed that the isolates were distinctly separated based on their AG types. Pathogenicity and aggressiveness of the isolates to several hosts was determined. AG-4 isolates from either cotton or tobacco were the most aggressive on the hosts tested, while AG-2-1 isolates were of moderate aggressiveness and were not pathogenic on barley.

Control Efficacy of Phloretin Isolated from Apple Fruits Against Several Plant Diseases

In the course of a searching natural antifungal compounds from plant sources, we found that the methanol extract (<TEX>$3,000\;{\mu}g/ml$</TEX>) of Malus domestica fruits had potential of control against rice blast (Magnaporthe grisea) and tomato late blight (Phytophthora infestans). Under bioassay-guided purification, we isolated phloretin, a phenolic compound, with in vivo antifungal activity against M. grisea. By 1-day protective application of phloretin (<TEX>$500\;{\mu}g/ml$</TEX>), the compound strongly inhibited the disease development of M. grisea and P. infestans on rice and tomato seedlings, respectively. And red pepper anthracnose caused by Colletotrichum coccodes also was moderately suppressed. However, rice sheath blight (Rhizoctonia solani AG1), and barley powdery mildew (Blumeria graminis f. sp. hordei) were hardly controlled. In addition, the compound showed in vitro antifungal activity against some plant pathogenic fungi including Phytophthora capsici, Alternaria panax, Sclerotinia sclerotiorum, R. solani AG4, and M. grisea. This is the first report on the antifungal activity of phloretin against plant pathogenic fungi.