Rhizoctonia Anastomosis Groups Research Articles

Diversity and pathogenicity of anastomosis groups of Rhizoctonia associated with brassicas in Brazil

Seventy-eight Rhizoctonia isolates were obtained from brassicas in five Brazilian states between 2015 and 2017. The isolates were identified by phylogenetic analysis of the rDNA internal transcribed spacer region (ITS) and tested for pathogenicity and aggressiveness. Sixty-eight isolates were identified as R. solani and 10 isolates as binucleate Rhizoctonia. The most prevalent anastomosis group (AG) was AG-4 HG-I (83.3% of isolates), followed by AG-A (7.7%), AG-1 IB (2.6%), AG-G (2.6%), AG-R (2.6%) and AG-2-2 IIIB (1.3%). AG-4 HG-I isolates were widely distributed and recovered from all Brazilian states sampled, including 12 brassicas-growing fields. All isolates were pathogenic to broccoli, cauliflower and kale, causing necrotic lesions on subterraneous stems. There was no significant difference in aggressiveness among the different AGs in broccoli, but AG-1 IB isolates were more aggressive to cauliflower and AG-4 HG-I isolates were more aggressive to kale. This study is the first report of AG-4 HG-I, AG-2-2 IIIB, AG-A, AG-G and AG-R infecting brassica plants in Brazil. To our knowledge, this is the first detailed research of the AG composition of Rhizoctonia populations causing brassica diseases in Brazil.

First Report of Binucleate Rhizoctonia AG-G Causing Grapevine (Vitis Vinifera) Trunk Diseases in California Nurseries.

Grapevine Trunk Diseases (GTD) are caused by a consortium of fungal pathogens that affect the biological functions of the vascular system of mature and young grapevines (Gramaje et al. 2018). We conducted surveys to profile GTD pathogens in California grapevine nurseries and collected 784 cuttings of cvs. Cabernet Sauvignon and Chardonnay grafted on 1103P rootstock. Several vines exhibited wood necrotic lesions and cankers at the graft union and the root ball (Figure 1A). Symptomatic wood tissues were cultured on PDA medium and after two weeks of incubation at room temperature (22°C), several known GTD pathogens were recovered. We also identified Rhizoctonia from 42 of the 784 vines (5.3% incidence) based on the morphological characteristics of a brown pigmented mycelium (Figure 1B), hyphae branched at a right angle with constrictions at the branch point (Figure 1C) and absence of spores (González García et al., 2006). A subsample of four isolates (DCHG2B, DCSG22R, JCSG9B, and JCHG12B) were randomly selected for further DNA-based taxonomic identification and pathogenicity evaluation to grapevine. The ITS and beta tubulin regions were amplified using the ITS1/ITS4 and B36F/B12R primer sets, respectively (González et al. 2006), and sequences were deposited in the NCBI database (Accession numbers: OR052655, OR052656, OR052657, OR052658 and OR059207, OR059208, OR059209, OR059210). Sequences displayed >99% and >96% identity with the respective ITS and beta tubulin sequences of the binucleate Rhizoctonia AG-G specimen C-653 (González et al. 2006). A phylogenetic tree constructed using the Neighbor-Joining method indicated a 100% bootstrap support with the binucleate Rhizoctonia AG-G (Figure 2). Pathogenicity of the binucleate AG-G Rhizoctonia were confirmed on two separate technical replicates using standard methods. For each replicate, one-year-old rootstock 1103P were wounded with sterile drill bits and inoculated with a single 5 mm diameter agar plug collected from Rhizoctonia growing cultures, while control vines were inoculated with sterile agar. The first replicate lasted 28 weeks with (DCHG2B, DCSG22R) inoculated on seven vines. The second bioassay lasted 24 weeks with two additional isolates (JCSG9B, JCHG12B) inoculated on twelve vines. Rhizoctonia-inoculated vines developed wood symptoms similar to those observed on cuttings in nurseries, with necrotic lesions lengths significantly longer than the controls (First replicate: 3.5  0.4 cm vs. 1.3  0.6 cm; Second replicate: 6.8  0.8 cm vs. 1.1  0.2 cm), based on one-way ANOVA statistical test (P value < 0.05). Rhizoctonia isolates recovery from wood necrotic lesions were confirmed by ITS sequencing, thereby fulfilling Koch's postulate. Several binucleate Rhizoctonia anastomosis groups, including AG-G, have been found to cause root rot and stem necrosis in plant nurseries (Aiello et al., 2017; Rinehart et al., 2007). Rhizoctonia has also been reported to be associated with grapevine nurseries in Europe (Pintos et al., 2018), South Africa (Halleen et al., 2003) and Australia (Walker, 1992). However, the multinucleate Rhizoctonia solani was the only species confirmed to cause root rot on grapevine (Walker, 1992). Our data suggests that the binucleate Rhizoctonia from the AG-G anastomosis group also cause wood necrosis in grapevine. Those findings warrant further studies on the complexity of Rhizoctonia anastomosis groups in nursery and their aggressiveness to grapevine.

Anastomosis Grouping and Phylogenetic analysis of Rhizoctonia Isolates on Wheat in Türkiye

The aim of this study is to determine the species and to evaluate the genetic diversity of the pathogenic and nonpathogenic Rhizoctonia spp. and anastomosis groups (AG) from wheat plants and rhizosphere soils in Turkey. Rhizoctonia species were isolated from plants and rhizosphere soils in wheat fields in 5 provinces in the Central Anatolian Region of Türkiye. As a result of the isolations, a total of 88 multinucleate (MN) and binucleate (BN) Rhizoctonia isolates were obtained. Identifications of the isolates were determined by rDNA-ITS sequence analyzes. The identified isolates belonged to MN Waitea circinata var. zeae, W. circinata var. oryzae, W. circinata var. circinata, MN Rhizoctonia solani AG 2-1, 2-2, 3, 4 HGII, 4 HGIII, 5, 8, 11 and BN AG A, DI, E, G, H, I, I-like, K. The most isolated group was W. circinata var. circinata. In pathogenicity studies, the most virulent group was determined as R. solani AG 4. Among the binucleate isolates, groups other than R. cerealis AG DI were not found to be pathogenic. Neighbor-joining phylogenetic trees of isolates were constructed from rDNA-ITS sequences. As a result of this study, the regional distribution of MN and BN Rhizoctonia AG isolates in important wheat production areas in the Central Anatolia Region, Turkey was determined. In addition, this study is the first comprehensive study in which the genetic diversity of Rhizoctonia AGs isolates obtained from wheat and rhizosphere soils in the region was evaluated with a molecular approach.

First Report of Binucleate Rhizoctonia AG-G Causing Root Rot of Japanese Bay Tree (Machilus thunbergii) in Korea.

Machilus thunbergii Sieb. & Zucc., commonly known as Japanese bay tree, is a large evergreen tree belonging to the Lauraceae family and is widely distributed in Asia, including Korea in subtropical and tropical forest areas (Wu et al., 2006). In April 2021, a root rot disease of 2-year-old Japanese bay trees was observed in a nursery on Wando Island in Korea. Tree roots exhibited brown to black discoloration, root rot, and deterioration, and leaves were severely wilted followed by plant death, with a disease incidence of approximately 30%. Symptomatic roots were surface sterilized with 1% NaOCl for 5 min and washed three times with distilled water. The root tissues were dried and plated on potato dextrose agar (PDA) and vegetable juice agar (V8) media. After 3-4 days of incubation at 25 ˚C, brown Rhizoctonia fungal-like colonies grew on both culture media. Hyphae of two representative isolates (CMML21-35 and CMML21-36) exhibited typical characteristics of Rhizoctonia, including a constriction of branching hyphae (Alvarez et al., 2013). In addition, two nuclei in each mycelial cell were observed after staining of mycelia with 0.1% Safranin O. The two isolates were identified as binucleate Rhizoctonia based on the microscopic observation. To confirm identification of the isolates, the internal transcribed spacer (ITS) and large subunit (LSU) regions were sequenced using two primer sets, ITS1/ITS4 and LROR/LR5 (White et al., 1990; Vilgalys and Hester 1990). BLASTn search analysis revealed that the ITS sequence of isolates had 99.66% (582 base pair matched of 584) sequence similarity with the sequences of binucleate Rhizoctonia (accession numbers JF519837 and AY927327, respectively) and the LSU sequence matched well with the sequence of Rhizoctonia sp. AG-G (accession number MN977413; similarity 99.56% and 910 base pair matched of 914). The sequences were deposited in GenBank under accession numbers OM049427 and OM049428 for ITS, OM679289 and OM679290 for LSU. Phylogenetic analysis of ITS and LSU regions revealed that the isolates grouped with binucleate Rhizoctonia anastomosis group AG-G (Teleomorph: Ceratobasidium sp.) with a high bootstrap value. Accordingly, the morphological and molecular characteristics confirmed the causal pathogen as binucleate Rhizoctonia AG-G (Jiang et al., 2016; Gonzalez et al. 2016). To test pathogenicity, a 2-year-old Japanese bay tree was inoculated by creating a hole in the soil near the root rhizosphere and placing 1.5g of ground mycelia obtained from a 5 day-old broth culture at two time points one week apart (Bartz et al., 2010). The control pot was inoculated with sterilized ddH2O. Inoculated and control plant pots were incubated in plastic boxes with 100% relative humidity at 25 ℃ for five days. After that, the pots were placed in the greenhouse at 23-25 ℃. One month post inoculation, initial disease symptoms were observed, and after two months, severe foliar wilting and eventual plant death occurred. The non-inoculated control remained healthy. The pathogen was re-isolated from infected roots, fulfilling Koch's postulates. The experiment was conducted three times with three replications. This is the first report of root rot of Japanese bay tree caused by binucleate Rhizoctonia AG-G in Korea and in the world. Previously, a pathogenic binucleate Rhizoctonia AG-G was isolated from colonized apple tree roots in orchards in Italy (Kelderer et al., 2012). The present study implies that this pathogen potentially causes a negative impact on the nursery and forest industries, thus further research on the screening for pathogenicity in other tropical and subtropical trees and also apple, which is an important crop in Korea, is needed.

Diversity and Aggressiveness of Rhizoctonia spp. from Nebraska on Soybean and Cross-Pathogenicity to Corn and Wheat.

Rhizoctonia and Rhizoctonia-like species of fungi that cause disease are known to have varying host ranges and aggressiveness. Accurate identification of these species causing disease is important for soybean disease management that relies upon crop rotation. The anamorphic genus Rhizoctonia contains several diverse species and anastomosis groups (AGs) including some known soybean pathogens, such as Rhizoctonia solani, whereas for others the ability to cause disease on soybean has not been well described. The present study was conducted to identify the predominant species and AG of Rhizoctonia from soybean, corn, and wheat fields that are pathogenic on soybean and characterize cross-pathogenicity to common rotational crops, corn and wheat. We surveyed for Rhizoctonia spp. in Nebraska; isolates were identified to species and AG, and aggressiveness was assessed. A total of 59 R.zeae isolates, 49 R.solani, nine binucleate Rhizoctonia, three R.circinata, and two R.oryzae isolates were collected in 2016 and 2017 from a total of 29 fields in 15 counties. The most abundant R.solani AGs were AG-4, AG-1 IB, AG-2-1, AG-3, and AG-5. R.solani AG-4 and R. zeae were found in all three regions of the state (west, central, and eastern). Some isolates that were most aggressive to soybean seedlings were cross-pathogenic on both wheat and corn. In addition, R. zeae was pathogenic on soybean when evaluated at 25°C, which is warmer than temperatures used previously, and isolates were identified that were aggressive on soybean and cross-pathogenic on both corn and wheat.

A Binucleate Rhizoctonia anastomosis group (AG-W) is the causal agent of sugar beet seedling damping-off disease in China

Two binucleate Rhizoctonia (BNR) isolates (HLJ-21 and HLJ-57) were recovered from diseased sugar beet seedlings in Heilongjiang Province, Northeastern China in May, 2015. The two isolates were identified as members of the anastomosis group (AG)-W based on morphological characteristics, hyphal anastomosis, polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP), and phylogenetic analysis of the internal transcribed spacer of the ribosomal DNA (rDNA ITS). The optimum temperature for hyphal growth was 25 °C and the mycelia retained a whitish appearance on potato dextrose agar as cultures aged. No sclerotia, however, were observed during the period of culture. Both of the isolates exhibited anastomosis with AG-W, but failed with other reference strains of BNRs. Restriction fragments resulting from the eight enzymes indicated that HLJ-21 and HLJ-57 isolates had the same restriction pattern as AG-W isolates. Phylogenetic analysis also indicated that the rDNA ITS region of the HLJ-21 and HLJ-57 isolates clustered with other AG-W isolates in the same clade. The AG-W isolates from sugar beet and potato were the causal agent of damping-off disease on sugar beet seedlings as well as root disease on wheat and corn seedlings. The AG-W isolates from sugar beet could also induce stem canker disease on potato plants. Notably, the isolates from sugar beet seedlings had a higher virulence on all of the tested crops than those isolates obtained from potato. This appears to be the first report of a AG-W BNR causing sugar beet seedling damping-off disease in China, and perhaps globally.

Diversity and pathogenicity of anastomosis groups of Rhizoctonia associated with potato stem canker and black scurf diseases in Brazil

Sixty-seven isolates of Rhizoctonia obtained from potato stems and tubers in the main potato growing regions of Brazil between 2013 and 2014 were analyzed for their molecular and pathogenicity characteristics. Using phylogenetic analysis of the internal transcribed spacer region of ribosomal DNA, 49 isolates were identified as R. solani and 18 isolates as binucleate Rhizoctonia (BNR). Twenty-five isolates (37.3%) belonged to anastomosis group (AG)-3 PT, 20 (29.9%) to AG-4 HGI, 15 (22.4%) to AG-R, three (4.5%) to AG-2-1, three (4.5%) to AG-A, and one (1.5%) to AG-1 IB. Most of the AG-3 PT, AG-4 HGI, AG-A and AG-R isolates, and all isolates of AG-2-1 and AG-1 IB were obtained from tubers with black scurf symptoms. AG-4 HGI isolates were widely distributed and recovered from six out of seven Brazilian states, including 11 potato-growing fields. All isolates were pathogenic to potato, causing necrotic lesions on subterraneous stems, but there were differences in aggressiveness among different AGs. AG-4 HGI was significantly more aggressive to potato plants than other AGs. To our knowledge, this is the first detailed report of the AG composition of Rhizoctonia populations causing potato diseases in different Brazilian regions.

Variation in Fungicide Sensitivity Among Rhizoctonia Isolates Recovered from Potatoes in South Africa

Rhizoctonia is a major pathogen of potato causing substantial yield losses worldwide. Control of Rhizoctonia diseases is based predominantly on the application of fungicides. However, little is known about the fungicide response variability of different Rhizoctonia anastomosis groups associated with potato diseases in South Africa. A total of 131 Rhizoctonia isolates were obtained from potato growing regions of South Africa from 2012 to 2014 and evaluated for sensitivity to fungicides in vitro and in vivo. The fungicides comprised six chemical formulations and one bio-fungicide representing seven Fungicide Resistance Action Committee groups. All Rhizoctonia anastomosis groups were sensitive to tolclofos-methyl (EC50: 0.001 to 0.098 μg a.i. ml-1) and fludioxonil (EC50: 0.06 to 0.09 μg a.i. ml-1) and showed variation in sensitivity to pencycuron, iprodione, benomyl, and Bacillus subtilis QST 713. However, for azoxystrobin, Rhizoctonia isolates exhibited variable sensitivity ranging from sensitivity (EC50: <0.09 μg a.i. ml-1) to insensitivity with EC50 values exceeding 5 μg a.i. ml-1. In greenhouse and field trials, tolclofos-methyl and fludioxonil exhibited significantly greater control of stem and black scurf whereas azoxystrobin was the least effective. This work demonstrated variable sensitivity within and among anastomosis groups of R. solani and binucleate Rhizoctonia to different fungicides. Information on fungicide sensitivity of Rhizoctonia isolates is crucial in the development of effective Rhizoctonia control strategies and facilitates monitoring of fungicide insensitive isolates in the pathogen population.

Characterization and pathogenicity of Rhizoctonia spp. isolated from winter squash in the Black Sea region of Turkey

A total of 27 isolates of Rhizoctonia spp. were recovered from diseased winter squash (Cucurbita maxima) plants, collected from Amasya, Ordu, Samsun and Sinop provinces in the Black Sea region, Turkey. The isolates of Rhizoctonia spp. associated with root and crown rot of winter squash were examined for their cultural characteristics, anastomosis groups and subgroups using sequence analyses of the rDNA-ITS and pathogenicity. Based on ITS1–5.8S-ITS2 rDNA sequencing, Rhizoctonia solani anastomosis group (AG) 4 was the pathogen most frequently recovered (42.8 %) in all fields surveyed. Twenty-four isolates of R. solani AG 4 were further divided into three subgroups, AG 4 HG-I (13), AG 4 HG-II (6) and AG 4 HG-III (5). In addition, three isolates recovered from different fields were identified as binucleate (BN) Rhizoctonia AG A (7.4 %) and AG K (3.7 %). All isolates grew at temperatures of 10, 15, 20, 25, 30 and 35 °C, and they were completely inhibited at 5 and 40 °C. The results of pathogenicity tests on winter squash seedlings showed statistically significant differences (P < 0.001) among the mean disease severity index (DSI) of three subgroup isolates of R. solani AG 4. The DSI of the isolates of R. solani AG 4 ranged from 2.0 to 5.0. Isolates of AG 4 HG-I and HG-III had the highest virulence levels (DSI ranged from 3.8 to 5.0). The virulence level of isolates of BN Rhizoctonia AG A and AG K were low (DSI ranged from 2.0 to 2.3). To our knowledge, this is the first report of R. solani AG 4 HG-I, HG-II, and HG-III, and BN Rhizoctonia AG A and AG K occurring on winter squash, one of the most important cucurbit species in Turkey.

MODELLING THE TEMPERATURE-MYCELIAL GROWTH RATE RELATIONSHIPS IN FUNGI RHIZOCTONIA spp.

Temperature is one of the most important factors affecting the fungal growth and development and their relationships have been investigated in physiological and epidemiological studies. Cardinal temperatures of fungi were obtained from these studies and they were mainly performed via visual estimation of polynomial modeling, which resulted in non-biologically meaningful parameters. In this study, eight nonlinear regression models were used for modeling temperature-mycelial growth rate relationships of three anastomosis groups of Rhizoctonia spp. (AG 1-IA, AG 4 and WAG-Z). Comparison and selection of the best-fitting model were conducted based on SE, adjusted R2, RMSE, a, and b, and they were then ranked by AIC statistic. The results indicated that the segmented model was the best fit for all three AGs. Based on AICc and i, the segmented model was the best fit model for describing growth rate of AG 4 and WAG-Z at different temperatures. In case of AG 1-IA, the cubic model could produce the least amount of AICc and the segmented model ranked second. Besides, i of dent-like model was <10 in all three cases and, therefore there was not a significant difference in fitness of these models. Estimated Tb and (Tc) of AG 1-IA, AG 4 and WAG-Z by the segmented model were 7.94, (38.69)°C, 7.66, (39.59)°C and 8.11, (41)°C, respectively. Optimum temperatures estimated by the segmented model for AG 1-IA, AG 4 and WAG-Z were 28.58°C, 29.99°C and 33.72°C, respectively.

Molecular Characterization, Morphological Characteristics, Virulence, and Geographic Distribution of Rhizoctonia spp. in Washington State.

Rhizoctonia root rot and bare patch, caused by Rhizoctonia solani anastomosis group (AG)-8 and R. oryzae, are chronic and important yield-limiting diseases of wheat and barley in the Inland Pacific Northwest (PNW) of the United States. Major gaps remain in our understanding of the epidemiology of these diseases, in part because multiple Rhizoctonia AGs and species can be isolated from the same cereal roots from the field, contributing to the challenge of identifying the causal agents correctly. In this study, a collection totaling 498 isolates of Rhizoctonia was assembled from surveys conducted from 2000 to 2009, 2010, and 2011 over a wide range of cereal production fields throughout Washington State in the PNW. To determine the identity of the isolates, PCR with AG- or species-specific primers and/or DNA sequence analysis of the internal transcribed spacers was performed. R. solani AG-2-1, AG-8, AG-10, AG-3, AG-4, and AG-11 comprised 157 (32%), 70 (14%), 21 (4%), 20 (4%), 1 (0.2%), and 1 (0.2%), respectively, of the total isolates. AG-I-like binucleate Rhizoctonia sp. comprised 44 (9%) of the total; and 53 (11%), 80 (16%), and 51 (10%) were identified as R. oryzae genotypes I, II, and III, respectively. Isolates of AG-2-1, the dominant Rhizoctonia, occurred in all six agronomic zones defined by annual precipitation and temperature within the region sampled. Isolates of AG-8 also were cosmopolitan in their distribution but the frequency of isolation varied among years, and they were most abundant in zones of low and moderate precipitation. R. oryzae was cosmopolitan, and collectively the three genotypes comprised 37% of the isolates. Only isolates of R. solani AG-8 and R. oryzae genotypes II and III (but not genotype I) caused symptoms typically associated with Rhizoctonia root rot and bare patch of wheat. Isolates of AG-2-1 caused only mild root rot and AG-I-like binucleate isolates and members of groups AG-3, AG-4, and AG-11 showed only slight or no discoloration of the roots. However, all isolates of AG-2-1 caused severe damping-off of canola, resulting in 100% mortality. Isolates of Rhizoctonia AG-8, AG-2-1, AG-10, AG-I-like binucleate Rhizoctonia, and R. oryzae genotypes I, II, and III could be distinguished by colony morphology on potato dextrose agar, by PCR with specific primers, or by the type and severity of disease on wheat and canola seedlings, and results of these approaches correlated completely. Based on cultured isolates, we also identified the geographic distribution of all of these Rhizoctonia isolates in cereal-based production systems throughout Washington State.

Rhizoctonia Anastomosis Groups Associated with Diseased Rooibos Seedlings and the Potential of Compost as Soil Amendment for Disease Suppression.

Rhizoctonia spp. associated with rooibos in the Western Cape province of South Africa were recovered during the 2008 season by planting seedlings in rhizosphere soils collected from 14 rooibos nurseries. In all, 75 Rhizoctonia isolates were obtained, of which 67 were multinucleate and 8 were binucleate Rhizoctonia spp. The identity of these isolates to anastomosis group (AG) was determined through sequence analysis of the ribosomal DNA internal transcribed spacer region. The collection of multinucleate isolates included representatives of AG-2-2 (67%), AG-4 HGI (14%), AG-11 (5%), and R. zeae (3%). Binucleate AGs included AG-Bo (4%) and AG-K (4%) and an unidentified binucleate Rhizoctonia (3%). Rhizoctonia solani AG-2-2 was the most widely distributed species of Rhizoctonia detected among the 11 nurseries sampled. All AGs recovered from rooibos have been previously reported on crop plants in South Africa, with the exception of R. zeae. However, this is the first study to classify the Rhizoctonia AGs recovered from rooibos. In glasshouse bioassays, the most virulent Rhizoctonia AGs on rooibos and lupin were AG-2-2, AG-4 HGI, and AG-11. Although plant damage was less than that observed for lupin and rooibos, oat was significantly affected by AG-2-2 and AG-4 HGI. Two composts sourced from independent suppliers were evaluated for disease suppression under glasshouse conditions. Compost amendment suppressed damping-off by most R. solani AGs, except for AG-4 HGI. Furthermore, within AG-2-2, suppression by compost was isolate specific.

Characterization and colonization of endomycorrhizal Rhizoctonia fungi in the medicinal herb Anoectochilus formosanus (Orchidaceae).

The medicinal effects and techniques for cultivating Anoectochilus formosanus are well-documented, but little is known about the mycorrhizal fungi associated with A. formosanus. Rhizoctonia (Thanatephorus) anastomosis group 6 (AG-6) was the most common species isolated from fungal pelotons in native A. formosanus and represented 67 % of the sample. Rhizoctonia (Ceratobasidium) AG-G, P, and R were also isolated and represent the first occurrence in the Orchidaceae. Isolates of AG-6, AG-R, and AG-P in clade I increased seed germination 44–91 % and promoted protocorm growth from phases III to VI compared to asymbiotic treatments and isolates of AG-G in clade II and Tulasnella species in clade III. All isolates in clades I to III formed fungal pelotons in tissue-cultured seedlings of A. formosanus, which exhibited significantly greater growth than nonmycorrhizal seedlings. An analysis of the relative effect of treatment ( {widehat{p}}_i ) showed that the low level of colonization ( {widehat{p}}_i = 0.30hbox{--} 0.47 ) by isolates in clade I resulted in a significant increase in seedling growth compared to isolates in clades II (0.63–0.82) and III (0.63–0.75). There was also a negative correlation (r = −0.8801) with fresh plant weight and fungal colonization. Our results suggest that isolates in clade I may represent an important group associated with native populations of A. formosanus and can vary in their ability to establish a symbiotic association with A. formosanus. The results presented here are potentially useful for advancing research on the medicinal properties, production, and conservation of A. formosanus in diverse ecosystems.Electronic supplementary materialThe online version of this article (doi:10.1007/s00572-014-0616-1) contains supplementary material, which is available to authorized users.

AFLP Fingerprinting for Identification of Infra-Species Groups of Rhizoctonia solani and Waitea circinata

Patch diseases caused by Thanatephorus cucumeris (Frank) Donk and Waitea circinata Warcup and Talbot varieties (anamorphs: Rhizoctonia species) pose a serious threat to successful maintenance of several important turfgrass species. Reliance on field symptoms to identify Rhizoctonia causal agents can be difficult and misleading. Different Rhizoctonia species and Anastomosis Groups (AGs) vary in sensitivity to commonly applied fungicides and they also have different temperature ranges conducive for causing disease. Thus correct identification of the causal pathogen is important to predict disease progression and make future disease management decisions. Grouping Rhizoctonia species by anastomosis reactions is difficult and time consuming. Identification of Rhizoctonia isolates by sequencing Internal Transcribed Spacer (ITS) region can be cost prohibitive. Some Rhizoctonia isolates are difficult to sequence due to polymorphism of the ITS region. Amplified Fragment Length Polymorphism (AFLP) is a reliable and cost effective fingerprinting method for investigating genetic diversity of many organisms. No detailed analyses have been done to determine the suitability of AFLP for inferring infra-species level of Rhizoctonia isolates. The objective of the present study was to develop AFLP fingerprinting to identify infra-species level of unknown R. solani Kuhn and W. circinata isolates. Seventy-nine previously characterized R. solani (n=55) and W. circinata (n=24) isolates were analyzed with AFLP markers generated by four primer pairs. Unweighted Pair Group Method with Arithmetic Mean (UPGMA) correctly grouped R. solani and W.circinata isolates according to their AG, AG subgroup or W.circinata variety. Principle component analysis (PCA) corroborated UPGMA clusters. To our knowledge this is the first time AFLP analysis has been tested as a method to decipher the AG, AG subgroup or W.circinata variety across a wide range of Rhizoctonia isolates.

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.

Characterization and Sensitivity to Fungicides of Rhizoctonia spp. Recovered from Potato Plants in Bolu, Turkey

AbstractIsolates of Rhizoctonia spp. associated with stem canker and black scurf disease of potato were examined for their anastomosis group, sequence variations in the ITS‐5.8S rDNA region, pathogenicity and sensitivity to fungicides. A total of 92 isolates were obtained from diseased tuber, stolon and sprouts of the potato plants, collected from five districts of Bolu province, Turkey. Based on the anastomosis group and the similarity of the nucleotide sequence of the ITS‐5.8S rDNA, most of the isolates (81.5%) were identified as AG 3 PT. Other isolates belonged to AG 2‐1 (1.08%), AG 2‐2 IV (1.08%), AG 4 HG II (8.07%), AG 5 (2.17%), binucleate Rhizoctonia AG A (1.08%) and AG K (4.35%). Pathogenicity tests showed that isolates of AG 3 PT, AG 4 HG II and AG 5 caused similar degrees of disease severity on 45‐day‐old potato seedlings, whereas AG 2‐1 was moderately virulent. AG 2‐2 IV and binucleate Rhizoctonia spp. were weakly pathogenic or non‐pathogenic on potato seedlings. In this study, anastomosis groups of Rhizoctonia spp. isolates associated with potato in Turkey were characterized for the first time using molecular techniques and classified at the level of subgroups. Furthermore, the effect of selected fungicides was evaluated on disease development caused by soil‐borne inoculums of different anastomosis groups (AGs). Flutolanil and Bacillus subtilis QST 713 were found to be most effective against the Rhizoctonia isolates tested. These results revealed significant differences among the fungicides on disease development resulted from the different AGs.

English

The interaction of binucleate Rhizoctonia (BNR) anastomosis group (AG)-A isolate W7, Rhizoctonia solani AG-4 and cucumber seedlings were investigated to elucidate the mechanism of biocontrol of Rhizoctonia solani by BNR. Hypocotyls of Cucumis sativus L. cv. Jibai were inoculated with a virulent isolate of R. solani AG-4 isolate C4 and examined with light microscopy and scanning and transmission electron microscopy. The hyphae of the virulent isolate colonized the outer surface of the hypocotyl and penetrated the epidermal and cortical cells to the pith of hypocotyls. The hyphae of a non-pathogenic species of BNR isolate W7 did not penetrate the cortical cells but instead colonized the outer epidermal cells 12 h after inoculation. Accumulation of mucilage occurred on the surface after dense colonization of the hypocotyls surface by the BNR. Observation of the hypocotyls surface pre-inoculated with BNR and challenged inoculated with R. solani showed constricted and deformed hyphae of R. solani , which were prevented to penetrate the epidermal cells. Pre-inoculating of BNR isolate caused profused formation of the mucilage which lysed BNR hyphae and restricted the growth of R. solani . The mucilage was identified as pectic substances. Analysis of pectin contents from dried hypocotyls tissues showed highly methylated pectin with low uronic acid contents in the seedlings inoculated with R. solani and unprotected by BNR while BNR- treated seedlings showed less methylated pectin and high amount of uronic acid. Furthermore, the BNR-inoculated seedlings showed significant ( P = 0.05) increase in calcium, indicating high amount of wall-bound cations in cell walls. Pectic substances accumulation and increased calcium in the cucumber tissues greatly contributed in the protection of cucumber seedlings against Rhizoctonia damping off by a non-pathogenic species of Rhizoctonia. Keywords: Rhizoctonia solani , binucleate Rhizoctonia , cucumber, biological control African Journal of Biotechnology , Vol. 13(5), pp. 640-650, 29 January, 2014

Survey on the prevalence of Rhizoctonia spp. in European soils and determination of the baseline sensitivity towards sedaxane

The prevalence of Rhizoctonia spp. in European soils was determined by analysing soil samples from 282 locations. Rhizoctonia spp. were found in 68% of these samples from France, Germany, the UK, Poland, Italy, Spain, Hungary and the Czech Republic. Samples from 136 locations were further analysed by pyrosequencing. Seventy‐six percent of the isolates were Rhizoctonia solani and 24% binucleate Rhizoctonia spp. Rhizoctonia solani anastomosis group (AG) 5 was detected most frequently (25%), followed by AG 9 (16%) and AG 4 (13%). For the binucleate Rhizoctonia spp., AG E was most prevalent (13%). Rhizoctonia cerealis was not detected in soil samples. Soil type or cropping history had no effect on the type of Rhizoctonia observed. Rhizoctonia solani AG 5 was the most frequently detected AG irrespective of the previous crop. The spectrum of AGs detected was similar for France, Germany and Poland but was significantly different for the UK (P = 0·0016). Finally, the baseline sensitivity towards sedaxane, a new active ingredient for seed treatment, was analysed for all isolates. The results indicate a low baseline sensitivity (average EC50 of 0·028 p.p.m.) for all Rhizoctonia AGs. No difference in sensitivity was observed with the isolates obtained from different countries.

Molecular diversity of binucleate Rhizoctonia AG-A in China

AG-A belongs to the binucleate Rhizoctonia (BNR) anastomosis group (AG) of the Ceratobasidium teleomorph, which parasitizes the roots of many plant species. Ninety nine isolate species of AG-A were obtained from Tibet, Sichuan, and Yunnan Province in China. All isolates were divided into three types based on their cultural characteristics. Type I: abundant aerial mycelia, dense hyphae, loose sclerotia; Type II: abundant aerial mycelia, no sclerotia. Type III: sparse aerial mycelium and no sclerotia. All of the isolates infected the seedlings of Chinese mustard and Chinese cabbage, causing the formation of lesions on the stem and a brown discoloration of the roots. Sequence analysis of the 5.8S rDNA-ITS showed a similarity of 98–100% among the isolates. Inter Simple Sequence Repeat (ISSR) was used to detect genetic variation in binucleate Rhizoctonia spp. Forty two AG-A isolates were amplified using 15 random primers. From a total of 164 bands, 144 bands (87.8%) were polymorphic in the 42 tested isolates. A dendrogram showing genetic relationships between the isolates was constructed using unweighted pair-group averages based on genetic distances. According to the dendrogram, the 42 tested isolates could be aligned into three clusters with a genetic similarity coefficient of 0.29, the first clusters including 27 isolates with III of culture characteristics on PDA; the second clusters included eight isolates with I of cultural characteristics on PDA; the third cluster included seven isolates with II of cultural characteristics on PDA. The results of ISSR analysis showed an association between the hosts of these isolates. Our results showed that ISSR analysis can reveal more molecular variation among isolates of AG-A than sequence analysis using the 5.8S rDNA-ITS.

Seasonal Prevalence of Species of Binucleate Rhizoctonia Fungi in Growing Medium, Leaf Litter, and Stems of Container-Grown Azalea.

Rhizoctonia web blight is an annual problem on container-grown azalea (Rhododendron spp.) in the southern and eastern United States but little is documented about the distribution or persistence of Rhizoctonia spp. in container-grown azalea. Sixty web-blight-damaged azalea plants ('Gumpo White') were collected in August 2005 and 2006 and arranged in a completely randomized design on an outdoor irrigation pad. A nylon mesh bag containing 30 necrotic leaves collected from web-blight-damaged 'Gumpo White' azalea plants were placed on the surface of the medium under the plant canopy in each container to simulate leaf litter. Ten plants were destructively sampled into eight zones by dividing stems into three zones (lengths of 0 to 2, 4 to 6, and 9 to 15 cm above the medium surface), bagged leaves into one leaf litter zone, and the medium into four zones (three horizontal layers: 1 to 3, 3 to 7, and 7 to 10 cm below the medium surface, with the middle layer further divided by removing the central 7.5-cm-diameter core) in December, February, and May. Only the three stem zones were sampled from 10 plants in early and late June and late July. Of 8,940 total isolations, 3,655 fungi with morphological characteristics of a Rhizoctonia sp. were recovered. Percent recovery differed from the eight zones (P < 0.0001) but did not differ between years (P = 0.3950) and sampling times (P = 0.1896). Frequency of recovery of Rhizoctonia spp. was highest from the lower stem and the leaf litter, and decreased with distance from the leaf litter. Recovery from stems over the six sample times was analyzed separately. Percent recovery differed between stem zones (P < 0.0001), sample times (P = 0.0478), and experiment years (P < 0.0001). In both years, mean recovery of Rhizoctonia spp. was higher from the lower stem and decreased with distance to the upper stem layer. From a subsample of 145 isolates, 95.1% were identified as binucleate Rhizoctonia (BNR) anastomosis groups (AGs)-A, -G, -K, -R, -S, and -U (-P), and 2.8 and 2.1% were Rhizoctonia solani AG-2 and an uncultured Laetisaria sp., respectively. Based on frequency analysis, recovery of BNR AGs differed by plant zone (P < 0.0001) but not over sample times (P = 0.4831). The six AGs of BNR are the predominant Rhizoctonia fungi occupying the habitat niches in container-grown azalea, with little change in population frequency and composition from fall to summer; thus, BNR pathogenic and nonpathogenic to azalea have established a mixed Rhizoctonia community on container-grown azalea.