Binucleate Rhizoctonia Anastomosis Group Research Articles

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.

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.

Anastomosis Groups of Rhizoctonia solani and Binucleate Rhizoctonia Associated with Potatoes in Idaho.

A survey of the relative incidence of anastomosis groups (AGs) of Rhizoctonia spp. associated with potato disease was conducted in Idaho, the leading potato producing state in the U.S.A. In total, 169 isolates of Rhizoctonia solani and seven binucleate Rhizoctonia (BNR) isolates were recovered from diseased potato plants. The AG of each isolate was determined through real-time PCR assays for AG 3-PT and phylogenetic analysis of the internal transcribed spacer region of ribosomal DNA. AG 3-PT was the predominant AG, accounting for 85% of isolates recovered, followed by AG 2-1 (5.7%) and AG 4 HG-II (4.5%). Two different subsets of AG 2-1 isolates were recovered (subset 2 and 3). Three isolates each of AG A and AG K were recovered, as well as one isolate each of AG 5 and AG W. An experiment carried out under greenhouse conditions with representative isolates of the different AGs recovered from Idaho potatoes showed differences in aggressiveness between AGs to potato stems, with AG 3-PT being the most aggressive followed by an isolate of AG 2-1 (subset 3). The three BNR isolates representative of AG A, AG K, and AG W appeared to be less aggressive to potato stems than the R. solani isolates except for the AG 2-1 (subset 2) isolate. This is the first comprehensive study of the relative incidences of Rhizoctonia species associated with Idaho potatoes and the first study to report the presence of BNR AG W outside of China.

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.

Anastomosis Groups and Pathogenicity of Rhizoctonia solani and Binucleate Rhizoctonia from Potato in South Africa.

A survey of anastomosis groups (AG) of Rhizoctonia spp. associated with potato diseases was conducted in South Africa. In total, 112 Rhizoctonia solani and 19 binucleate Rhizoctonia (BNR) isolates were recovered from diseased potato plants, characterized for AG and pathogenicity. The AG identity of the isolates was confirmed using phylogenetic analysis of the internal transcribed spacer region of ribosomal DNA. R. solani isolates recovered belonged to AG 3-PT, AG 2-2IIIB, AG 4HG-I, AG 4HG-III, and AG 5, while BNR isolates belonged to AG A and AG R, with frequencies of 74, 6.1, 2.3, 2.3, 0.8, 12.2, and 2.3%, respectively. R. solani AG 3-PT was the most predominant AG and occurred in all the potato-growing regions sampled, whereas the other AG occurred in distinct locations. Different AG grouped into distinct clades, with high maximum parsimony and maximum-likelihood bootstrap support for both R. solani and BNR. An experiment under greenhouse conditions with representative isolates from different AG showed differences in aggressiveness between and within AG. Isolates of AG 2-2IIIB, AG 4HG-III, and AG R were the most aggressive in causing stem canker while AG 3-PT, AG 5, and AG R caused black scurf. This is the first comprehensive survey of R. solani and BNR on potato in South Africa using a molecular-based approach. This is the first report of R. solani AG 2-2IIIB and AG 4 HG-I causing stem and stolon canker and BNR AG A and AG R causing stem canker and black scurf on potato in South Africa.

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.

Anastomosis groups and pathogenicity of binucleate Rhizoctonia isolates associated with stem canker of potato in China

Potato stem canker caused by Rhizoctonia solani commonly occurs in potato-growing regions around the world, but little is known about whether binucleate Rhizoctonia (BNR) can incite this disease on potato plants in China. In the present study, a total of 69 BNR isolates were recovered from cankered subterraneous stems of potato collected from 17 provinces, autonomous regions, and municipalities of China. Based on the morphological characteristics and sequence analysis of the internal transcribed spacers of the ribosomal DNA, 48 isolates were classified as anastomosis group (AG)-A with a ratio of 69.56 %, 15 isolates (21.74 %) were AG-K, four isolates (5.80 %) were AG-F, one isolate (1.45 %) was AG-I and one isolate (1.45 %) was AG-U. Pathogenicity tests under glasshouse conditions revealed that all BNR isolates, except for the AG-I isolate, could induce symptoms of stem canker on potato plants with disease incidence ranging from 5.26 to 55.56 % and disease index ranging from 1.32 to 13.89, respectively. To our knowledge, this is the first detailed report about BNR AGs causing stem canker on potato plants in China.

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.

First Report of Root Rot Caused by Binucleate Rhizoctonia Anastomosis Group F on Musa spp.

A research program was initiated at the University of Georgia in 2003 to identify banana cultivars suitable for production in the coastal and southern areas of the state. During a root disease survey conducted in October 2007 on bananas (Musa spp.) grown at the University of Georgia Bamboo Farm and Coastal Gardens in Savannah, GA, root lesions and root rot were observed on banana cvs. Gold Finger, Kandarian, and Manzano. Root lesions were dark brown to black and irregular in shape, with partial or entire roots affected. Lateral roots and outer layers of cord roots (roots arising from interior layers of the corm) of infected plants were blackened and rotted. Diseased root samples were collected from three plants of each cultivar, surface sterilized with 0.6% sodium hypochlorite, and placed on tannic acid benomyl agar (TABA). Pure cultures of the fungus consistently associated with diseased tissue were obtained by subculturing hyphal tips on TABA. Mycelia of the fungus on potato dextrose agar (PDA) were light to deep brown and the hyphae tended to branch at right angles. A septum was present in each hyphal branch near the point of origin and a slight constriction at the branch was observed. The hyphae of two isolates were stained with 0.6% phenosafranin and 3% KOH and binucleate hyphal cells were observed. On the basis of these morphological features, the isolates appeared to be binucleate Rhizoctonia anamorphs (teleomorph Ceratobasidium Rogers). For molecular identification, the internal transcribed spacer (ITS) regions and the 5.8S gene from rDNA of the isolates were cloned and sequenced (GenBank Accession No. HQ168370). The ITS regions (775 bp) were 100% identical between the two isolates and 99% identical to Ceratobasidium sp. AG-F strain SIR-1 isolated from sweet potato in Japan (GenBank Accession No. AF354085). The anastomosis group of the isolates was confirmed by pairing with strain SIR-1 on PDA. On the basis of morphological and molecular characteristics and the anastomosis assay, the two isolates were identified as a Ceratobasidium sp. AG-F (1-3). Pathogenicity assays were conducted by inoculating banana plants (cv. Golden pillow, synonym = Manzano) grown in pots under greenhouse conditions (25 to 27°C). Twenty wheat seeds infested with each isolate were placed uniformly around each plant at a depth of 10 cm in the soil. The plants were incubated in the greenhouse and the roots were examined 2 months after inoculation. Brown-to-black lesions and root rot, identical to symptoms associated with field banana roots, were observed on all inoculated plants but not on the noninoculated control plants. The fungus was reisolated from affected root samples and the identity was confirmed by morphological and molecular characteristics and the anastomosis assay. To our knowledge, this is the first report of banana root rot caused by binucleate Rhizoctonia anastomosis group F. With the increased interest in producing bananas for food and ornamental purposes, the occurrence of Ceratobasidium root rot on bananas needs to be considered when designing disease management programs and searching for suitable cultivars for banana production.

Evaluation of strategies for the control of canola and lupin seedling diseases caused by Rhizoctonia anastomosis groups

Several methods with potential for the management of Rhizoctonia diseases of canola and lupin including plant resistance, fungicide seed treatment and biological control using binucleate Rhizoctonia anastomosis groups (AGs) were evaluated under glasshouse conditions. Screening included the examination of resistance of eight canola and eight lupin cultivars/selections to damping-off and hypocotyl/root rot caused by the multinucleate Rhizoctonia solani AG-2-1, 2–2, 4 and 11. All canola cultivars were highly susceptible to AG-2-1, but Rocket, Spectrum and 44C11 were more resistant than the other cultivars. Spectrum and 44C73 were also more resistant to AG-4 than the other canola cultivars. On lupin, R. solani AG-2-2 and 4 were most virulent, and the cultivar Cedara 6150 and selection E16 were most resistant to AG-2-2; Cedara 6150, E16, Mandelup and Quilinock were more resistant to AG-4 than the other cultivars/selections. The Lupinus luteus selections, E80.1.1.2 and E82.1.1 were most susceptible to AG-2-2, 4 and 11. Seed treatment with the fungicides Cruiser OSR (a.i. difenconazole, fludioxonil, metalaxyl-M, thiamethoxam) and SA-combination (a.i. iprodione, metalaxyl, thiram) significantly increased survival of canola and lupin seedlings, decreased hypocotyl/root rot and improved the percentage of healthy seedlings, with the SA-combination being significantly more effective than Cruiser OSR. Application of the binucleate Rhizoctonia AGs (A, Bo, K and I) significantly increased the survival of lupin seedlings inoculated with R. solani AG-2-2 and 4, and AG-I and K significantly improved survival of canola in the presence of AG-4. This is the first report of the potential of binucleate AGs to protect canola and lupin seedlings against infection by multinucleate AGs.

Characterization of fungi (Fusarium and Rhizoctonia) and oomycetes (Phytophthora and Pythium) associated with apple orchards in South Africa

Several species of fungi and oomycetes including Fusarium, Rhizoctonia, Phytophthora and Pythium have been reported as root pathogens of apple where they contribute to a phenomenon known as apple replant disease. In South Africa, little is known about specific species in these genera and their pathogenicity toward apple. Therefore, these aspects were investigated along with the development and optimization of qPCR tests for detection and quantification of the most virulent oomycete species. In eight investigated orchards, the oomycete Phythophthora cactorum was widely distributed, while nine Pythium species were differentially distributed among the orchards. Pythium irregulare was the most widely distributed and the most virulent species along with P. sylvaticum, P. vexans and Ph. cactorum. Seven binucleate Rhizoctonia anastomosis groups (AGs) were also differentially distributed among the orchards, with the majority appearing to be non-pathogenic while certain AG-I and AG-F isolates exhibited low virulence on apple. In the genus Fusarium, F. oxysporum was widely distributed, but isolates were non-pathogenic. Fusarium solani and F. avenaceum were less frequently encountered, with only some isolates having low virulence. qPCR data obtained from seedling roots inoculated with the most virulent Pythium species (P. irregulare, P. sylvaticum and P. vexans) and the genus Phytophthora were not always reproducible between trials, or isolates of the same species. In general, seedling growth inhibition was associated with the presence of a low amount of pathogen DNA (±40 fg μl−1 to 2 pg μl−1) in roots. Pythium irregulare, although having the lowest DNA concentrations in roots, was the only species for which a significant negative correlation was found between seedling weight and pathogen DNA concentration.

Chemical and Hot Water Treatments to Control Rhizoctonia AG P Infesting Stem Cuttings of Azalea

In the southern and eastern United States, azalea stems cut during the spring for propagation may be infested with Rhizoctonia spp. Multiple methods were evaluated in a series of laboratory experiments for the purpose of eliminating Rhizoctonia from stem cuttings of Rhododendron L. ‘Gumpo White’ [‘Gumpo White’ (Satsuki) azalea] to prevent spread of azalea web blight during the propagation phase of nursery production. Leafless stem sections were inoculated with an isolate of binucleate Rhizoctonia anastomosis group P (AG P). Disinfestants (sodium hypochlorite, hydrogen dioxide, and quaternary ammonium chloride) or fungicides (chlorothalonil + thiophanate-methyl and flutolanil) applied at several rates (below, at, and above label rates) did not eliminate Rhizoctonia AG P from stem sections. Recovery of Rhizoctonia AG P was not reduced by submersing stem pieces in 45 °C water, but was eliminated at water temperatures of 50 °C or greater. Mortality of Rhizoctonia infesting azalea stem pieces was best explained by a cubic regression model. Mortality increased with increasing time (0, 1.5, 3, 4.5, 6, 7.5, 9, 10.5, 12, 15, 18, and 21 min) in water at 50 and 55 °C and with increasing temperatures (52, 55, 58, 61, 64, 67, and 70 °C) when stem pieces were submerged for 30 and 60 s. The duration of hot water treatment at which 99% of stem pieces were predicted to be free of Rhizoctonia was 20 min 16 s at 50 °C and 5 min 19 s at 55 °C. The average water temperature at which 99% of the stem pieces were predicted to be free of Rhizoctonia was 60.2 and 56.9 °C when stem pieces were submerged for 30 and 60 s, respectively. Only minor leaf damage occurred on terminal, leafy stem cuttings when submerged in 50 °C water after 40 min. Severe leaf damage did occur if cuttings were submerged long enough in water of 55 °C or greater. Leaf damage was predicted to exceed a proportional leaf damage value of 0.25 (indicating severe damage) when leafy stem cuttings were submerged in 55 °C water for longer than 13 min 54 s or for 30 and 60 s with water temperature greater than 57.4 and greater than 56.8 °C, respectively. Of the methods tested, submersion in hot water has the greatest potential for eliminating Rhizoctonia AG P from azalea stem cuttings. Submerging stem pieces in 50 °C water for 21 min eliminated Rhizoctonia and provided the least risk for development of severe leaf damage.

Phylogenetic relatedness of the M2 double-stranded RNA in Rhizoctonia fungi

Isolates from closely related fungi in the Rhizoctonia species complex were examined for the occurrence of the M2 double-stranded RNA (dsRNA) by amplifying a conserved 1000 nucleotide region of the dsRNA with reverse transcription PCR. The M2 dsRNA was detected in representative isolates belonging to three anastomosis groups (AG) of R. solani (AG-1-IA, AG-4 and AG-6; teleomorph = Thanatephorus) and four AGs of binucleate Rhizoctonia (AGA, AG-F, AG-R and AG-U; teleomorph = Ceratobasidium). Amplified PCR products from the 3′ region of the M2 dsRNA from a representative sample of 12 isolates from eight different AGs were sequenced and subjected to parsimony analysis and coalescent simulations to infer ancestral lineages and to reconstruct the ancestral history of haplotypes. Seven dsRNA haplotypes were inferred from the sample of 12 isolates. One haplotype was composed of only isolates of Ceratobasidium belonging to different AGs. The rooted gene genealogies from coalescent simulations suggested that the ancestral M2 dsRNA haplotype most likely evolved in Thanatephorus (anamorph = R. solani AG-1-IA) and has been acquired recently by isolates of Ceratobasidium. Reconstruction of the ancestral history of haplotypes with a parsimony-based approach that assumes both mutation and recombination suggested that four haplotypes recombined before coalescing to their most recent common ancestor, while three haplotypes coalesced without recombination in the recent past. There was no unique association of haplotype within a specific AG of either Ceratobasidium or Thanatephorus to support co-evolution of the M2 dsRNA within the fungal host. To our knowledge this is the first report of a dsRNA occurring in Ceratobasidium that also is present in Thanatephorus.

Characterization of isolates of binucleate Rhizoctonia spp. associated with strawberry black root rot complex using fatty acid methyl ester (FAME) profiles

Black root rot is an important disease of strawberry caused by a complex of fungi that includes species of Rhizoctonia. In this study, a modified MIDI method (Microbial Identification System) was investigated for its utility to differentiate isolates of the three different anastomosis groups (AGs) of binucleate Rhizoctonia spp., associated with strawberry black root rot complex representing AG-A, AG-G, and AG-I. A total of 11 fatty acids were detected, and the FAME profiles for isolates of the three different AGs of Rhizoctonia spp. varied quantitatively and qualitatively. Moreover, the modified MIDI method will be a useful discriminatory tool for fungal identification and classification of the AGs of binucleate Rhizoctonia spp. associated with strawberry black root rot complex.

Identification and Pathogenicity of Rhizoctonia spp. Causing Wirestem of Betula nigra in China

During July 2004, wirestem was frequently observed on the seedlings of Betula nigra at Dehong district in Yunnan Province, China. Isolates of Rhizoctonia spp. consistently obtained from their diseased leaves, roots and stems were identified as belonging to binucleate Rhizoctonia anastomosis groups (AG) AG-P and AG-R, and R. solani AG-I IB and AG-4 HG-I, based on cultural characteristics, nuclear staining, anastomisis reaction and analysis of their ITS rDNA region. The percentage of recovery of AG-P, AG-1, AG-R and AG-4 was 48%, 39%, 8% and 3%, respectively. This is the first report of wirestem of red birch cause by binucleate Rhizoctonia AG-P and AG-R, and R. solani AG-1 IB and AG-4 HG-I in China.

Analysis of whole cellular fatty acids and anastomosis relationships of binucleate Rhizoctonia spp. associated with Ceratobasidium cornigerum

Previous research has demonstrated that whole cellular fatty acids analysis is a useful tool for identifying and establishing taxonomic relationships between anastomosis groups (AGs) and related Rhizoctonia isolates. In this experiment, the composition of fatty acid of 28 isolates of teleomorph genus Ceratobasidium cornigerum, consisting of binucleate Rhizoctonia, AG-A, AG-B(o), AG-C, AG-P, and AG-Q, was evaluated using gas chromatography. Eleven fatty acids identified, i.e., myristic, pentadecanoic, palmitic, 2-hydroxypalmitic, palmitoleic, heptadecanoic, 9-heptadecenoic, stearic, oleic, linoleic, and linolenic acids, were present in isolates of AG-A, AG-B(o), AG-C, AG-P, and AG-Q. The major fatty acids, palmitic, oleic, and linoleic acids, were common in all isolates, constituting 87.1% to 94.7% of the whole cellular fatty acids identified. Isolates within the same AG were closely clustered, whereas isolates from different AGs were clearly and distinctly clustered based on average linkage cluster analysis of whole cellular fatty acids. Principal-component analysis generated from all fatty acids also confirmed the divergent separation of the 5 AGs of binucleate Rhizoctonia.

Effects of nematicides on cotton root mycobiota.

Baseline information on the diversity and population densities of fungi collected from soil debris and cotton (Gossypium hirsutum L.) roots was determined. Samples were collected from Tifton, GA, and Starkville, MS containing cotton field soil treated with the nematicides 1,3-dichloroproprene (fumigant) and aldicarb (granules). A total of 10,550 and 13,450 fungal isolates were collected from these two study sites, respectively. Of this total, 34 genera of plant pathogenic or saprophytic species were identified. Pathogenic root fungi included Fusarium spp. (40% of all isolations), Macrophomina, Pythium, Rhizoctonia, and Sclerotium. Fusarium and Rhizoctonia were the most common fungal species identified and included F. oxysporum, F. verticillioides and F. solani, the three Fusarium species pathogenic on cotton plants. Population densities of Fusarium were not significantly different among locations or tissue types sampled. Macrophomina was isolated at greater numbers near the end of the growing seasons. Anastomosis groups of R. solani isolated from roots and soil debris included AG-3, -4, -7, 2-2, and -13 and anastomosis groups of binucleate Rhizoctonia included CAG-2, -3, and -5. Occurrences and frequency of isolations among sampling dates were not consistent. Fluctuations in the frequency of isolation of Rhizoctonia did not correspond with changes in frequency of isolation of the biological control fungus, Trichoderma. When individual or pooled frequencies of the mycobiota were compared to nematicide treatments, no specific trends occurred between treatments, application methods or rates. Results from this study show that use of 1,3-D and aldicarb in cotton fields does not significantly impact plant pathogenic fungi or saprophytic fungal populations. Thus cotton producers need not adjust seedling disease control measures when these two nematicides are used.

Identification and pathogenicity ofRhizoctonia solani and binucleateRhizoctonia anastomosis groups isolated from forage legumes in Erzurum, Turkey

Three-hundred-twenty-five isolates ofRhizoctonia (215R. solani and 110 binucleateRhizoctonia) were obtained from roots and crowns of alfalfa, sainfoin and common vetch grown in Erzurum, Turkey. The isolates were assigned to five anastomosis groups (AG) ofR. solani (AG-2-1, AG-3, AG-4, AG-5, and AG-10) and two anastomosis groups of binucleateRhizoctonia (AG-I and AG-K). In pathogenicity tests on alfalfa, sainfoin and common vetch, the highest disease severities were caused by isolates of AG-4 and AG-5. Isolates of AG-10 and AG-I were not pathogenic on the three tested forage legumes, whereas isolates of AG-K on alfalfa and sainfoin, and of AG-2-1 on sainfoin, were moderately virulent. Alfalfa isolate AG-3 was moderately virulent on sainfoin. This is the first report ofR. solani AG-3, AG-5, AG-10 and binucleateRhizoctonia AG-I on alfalfa. In addition, all theR. solani and binucleateRhizoctonia groups isolated from sainfoin and common vetch were recovered from these crops for the first time in Turkey.

Differentiation of Rhizoctonia spp. Based on their antigenic properties

Antigenic properties and serological relationship was investigated in binucleate and multinucleate Rhizoctonia spp. isolates from strawberries soybean, alfalfa and potato plants from Serbia, from Spain, anastomosis group testers and in strawberry roots inoculated with binucleate Rhizoctonia AG A and AG I. Two polyclonal antisera, unabsorbed and cross absorbed, were used in dot-immunobinding assay for these investigations. Antisera were produced against mycelial antigens of two isolates, which belong to different anastomosis groups (AG) of binucleate Rhizoctonia - AG A and AG I. Both unabsorbed antisera reacted positively with all tested Rhizoctonia spp. isolates, and the reaction was absent with control isolates (Pythium sp. Agaricus sp. and Fusarium sp). The results prove a close serological relationship among Rhizoctonia spp. isolates, and diversity between Rhizoctonia spp. and isolates from different taxonomic groups. Also, both unabsorbed antisera reacted with higher intensity with closely related antigens (belonging to the same AG) than with ones from another AG of binucleate Rhizoctonia or R. solani (multinucleate Rhizoctonia). After cross absorption specificity of the antisera was enhanced, especially with the antiserum raised against mycelial proteins of binucleate Rhizoctonia AG I. This antiserum reacted positively only with antigens from the same AG, after cross absorption with antigens from AG A of binucleate Rhizoctonia and from R. solani AG 2-2. It proved to be specific to AG I of binucleate Rhizoctonia, and able to differentiate isolates of this AG from others. In this way the serological homology among isolates of one AG was proven, and also the diversity among isolates which belong to different AGs of binucleate Rhizoctonia as well as isolates of R. solani.

Grouping of Binucleate Rhizoctonia Anastomosis Group D (AG-D) Isolates into Subgroups I and II Based on Whole-Cell Fatty Acid Compositions

Grouping of Binucleate Rhizoctonia Anastomosis Group D (AG-D) Isolates into Subgroups I and II Based on Whole-Cell Fatty Acid Compositions