Characterization and Pathogenicity of Rhizoctonia spp. from Onion in Amasya, Turkey

Forty‐two isolates of Rhizoctonia spp. were obtained from onion in Amasya, Turkey. Of these, 29% were Rhizoctonia solani (AG‐4), 69% were Waitea circinata var. zeae (Rhizoctonia zeae) and 2% were binucleate Rhizoctonia (AG‐B). Most of the isolates were recovered from rhizosphere soil. In pathogenicity tests on onion, R. solani AG‐4 caused the greatest disease severity, those of W. circinata var. zeae were moderately virulent but binucleate Rhizoctonia isolates were of low virulence. This is the first report of binucleate Rhizoctonia AG‐B and W. circinata var. zeae occurring on onion in Turkey.     

Pectic zymogram variation and pathogenicity of <Emphasis Type="Italic">Rhizoctonia solani</Emphasis> AG-4 to bean (<Emphasis Type="Italic">Phaseolus vulgaris</Emphasis>) isolates in Isfahn,Iran

Rhizoctonia disease, caused by Rhizoctonia solani is one of the most important fungal diseases in bean fields in Isfahan, Iran. Bean plants showing stem and root cankers were collected and Rhizoctonia-like fungi obtained from the samples were identified by anastomosis. Pure cultures of bean isolates of R. solani were identified as AG-4. There were also AG-4 isolates from tomato, potato, cucumber, alfalfa and sugar beet in the areas sampled. A total of 163 isolates of R. solani AG-4 originating from stem and root cankers of beans were examined using pectic zymogram electrophoresis. Polygalacturonase (PG) and pectin estrase isozymes were observed in all AG-4 isolates tested. One (PG) and one pectic esterase (PE) band was found in common between all isolates examined. The electrophoretic patterns were grouped into seven zymogram groups (ZGs) according to the diagnostic PG and PE bands. One ZG occurred in a high frequency throughout the areas sampled. A pathogenicity test was conducted and representative isolates of each ZG were used to inoculate healthy bean plants. The results showed that each ZG caused different symptoms with varying severity. Isolates belonging to two ZGs were highly pathogenic causing root, stem and hypocotyl cankers whereas isolates of the other ZGs produced weak or no symptoms.     

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.     

Rhizoctonia spp. Causing Root and Hypocotyl Rot in Phaseolus vulgaris in Cuba

Sixty isolates of Rhizoctonia spp. were obtained from Cuban bean fields during the period 2004–2007. Isolates were characterized with different techniques, including nuclei staining, pectic zymogram, PCR–RFLP analysis of the rDNA–ITS region and sequencing of the rDNA–ITS region. The majority of the isolates were identified as multinucleate Rhizoctonia solani isolates, representing two different anastomosis groups (AGs), AG 2‐2 WB and AG 4 HGI; the remaining isolates were binucleate Rhizoctonia isolates and belonged to AG F and AG A. AG 4 HGI isolates were equally distributed in all soil types; AG 2‐2 isolates were more frequently isolated from cambisols, whereas AG F isolates were related to calcisols. Pathogenicity experiments in vitro and in the greenhouse, revealed that binucleate isolates only caused root rot, whereas R. solani isolates were able to cause root rot and hypocotyl rot. Furthermore, differences in virulence level were observed between R. solani and binucleate isolates and among different AGs. Isolates of R. solani AG 4 HGI and R. solani AG 2‐2 WB were the most aggressive, binucleate isolates of AG F were intermediate aggressive, whereas a binucleate isolate of AG A was weakly aggressive. In contrast with other reports about R. solani in bean, web blight symptoms were never observed during this study.     

Rhizoctonia spp. Associated with Container-Grown Ericaceous Plants in Scotland

Fungi with Rhizoctonia-like mycelia were isolated from the foliage, stem-base and roots of ericaceous plants collected from nurseries in Scotland. Isolated fungi were identified as either binucleate Rhizoctonia spp. or Rhizoctonia solani on the basis of hyphal characteristics and nuclear number. The optimum temperature range for growth of binucleate Rhizoctonia spp. and R. solani was 20 and 25 C, resepctively. All isolates tested for pathogenicity caused foliar browning, and webs of mycelial growth were observed on dead and dying foliage. Binucleate Rhizoctonia spp. and R. solani are recorded for the first time on container-grown ericaceous plants in Scotland.     

Interactions between cauliflower and Rhizoctonia anastomosis groups with different levels of aggressiveness

Background  

The soil borne fungus Rhizoctonia is one of the most important plant pathogenic fungi, with a wide host range and worldwide distribution. In cauliflower (Brassica oleracea var. botrytis), several anastomosis groups (AGs) including both multinucleate R. solani and binucleate Rhizoctonia species have been identified showing different levels of aggressiveness. The infection and colonization process of Rhizoctonia during pathogenic interactions is well described. In contrast, insights into processes during interactions with weak aggressive or non-pathogenic isolates are limited. In this study the interaction of cauliflower with seven R. solani AGs and one binucleate Rhizoctonia AG differing in aggressiveness, was compared. Using microscopic and histopathological techniques, the early steps of the infection process, the colonization process and several host responses were studied.     

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

Isolates 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.     

Biocontrol of Rhizoctonia crown and root rot of sugar beet by binucleate Rhizoctonia spp. and Laetisaria arvalis

Two isolates of Laetisaria arvalis and 10 of binucleate Rhizoctonia spp. (BNR) from the Ohio sugar beet production area, were tested in the greenhouse and field for biocontrol of Rhizoctonia crown and root rot of sugar beet, caused by Rhizoctonia solani anastomosis group 2, type 2. L. arvalis was ineffective in standard greenhouse tests, and the single isolate used in the field was generally ineffective. Seven of 10 BNR isolates effectively controlled crown and root rot in greenhouse tests. Delayed application of biocontrol agents to plants 5 – 10 wk old was generally more effective than applications made at planting. A BNR isolate significantly reduced % plant loss and disease ratings and increased yield in a 1985 field test as compared with the control infested with R. solani alone. Two BNR isolates were effective in a 1986 field test and increased yields c. 22% in comparison to a L. arvalis treatment, which did not differ from the R. solani-infested control. The Ohio binucleate Rhizoctonia isolates appear to have considerable potential as applied biocontrol agents and may play a role in the natural ecology of R. solani in the sugar beet production area of Ohio.     

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.     

Chemotaxonomy of fungi in the Rhizoctonia solani species complex performing GC/MS metabolite profiling

Hyphal anastomosis testing and molecular methods have been the primary criteria employed to understand the evolutionary and taxonomic relationships of the soil-borne fungal plant pathogen Rhizoctonia solani species complex. In this study, a metabolomics-based approach for characterizing and identifying isolates of R. solani using gas chromatography/mass spectrometry (GC/MS) metabolite profiling and footprinting was developed. Multivariate and hierarchical cluster analyses of GC/MS data provided resolution of isolates belonging to anastomosis groups (AGs) 1–6, 9, and 10 of R. solani. Clustering of R. solani AG-3 isolates, based on host origin, was also observed and attributed to metabolite-biomarkers belonging to amino, carboxylic and fatty acids. The chemotaxonomic approach using metabolomics is a high-throughput methodology that complements existing molecular approaches for the taxonomic investigation of Rhizoctonia isolates and monitoring of fungal metabolism.

     

Pathogenicity of Fusarium solani f. sp. glycines Isolates on Soybean and Green Bean Plants

Green bean plants were grown in a greenhouse in soil removed from a soybean field in 1996 that had a high incidence of soybean sudden death syndrome (SDS). Over a period of 4 weeks, isolations were made from taproot tissue of green bean plants to recover Fusarium isolates. Ten isolates of Fusarium solani were recovered and used to inoculate soybean and green bean plants in the greenhouse. These 10 isolates caused typical SDS symptoms on the soybean plants and caused a root and crown rot on green bean plants. The green bean plants did not develop typical symptoms associated with soybean SDS but, rather, leaves on infected plants showed yellowing and necrosis. Molecular data indicated that these 10 isolates were identical to Fusarium solani f. sp. glycines that cause soybean sudden death syndrome. All isolates were re-isolated from greenhouse-inoculated soybean and green bean plants.     

Molecular characterization of Rhizoctonia solani isolates the incitant of soybean root rot

Rhizoctonia solani isolates used in this investigation were identified as anastomosis-4 (AG-40), collected from different localities from Assiut governorate in Egypt. Pathogenicity test of seven isolates of R. solani was evaluated on soybean Giza 111 cultivar under greenhouse conditions. All tested isolates were able to infect soybean plants causing root rot with different degrees of severities, isolate No. 1, 2 and 3 showed significantly highest root rot severity, while isolate No. 5 gave the lowest percentage of root rot rating. The sodium dodecyl sulphate polyacrylamide gel electrophoresis patterns were used to compare three isolates of R. solani. There are no variations among R. solani isolates except a few exceptions according to their protein patterns. DNA markers obtained from all isolates showed genetic similarity among different isolates obtained from different geographical regions barring few exceptions. Correlation between DNA patterns of R. solani isolates and their virulence was detected, but no correlation with anastomosis groups (AG).     

Fungi and Gram-negative bacteria as soilborne minor pathogens of goat's rue (Galega orientalis Lam.)

Fungi and Gram-negative bacteria were isolated from inside the roots of field-grown goat's rue (Galega orientalis). Fungi were isolated from three plants out of a total of 45 tested. Two multinuclear Rhizoctonia solani isolates were identified to the anastomosis group 5 (R. solani AG-5-Gal) using pairings with known AG test cultures. One fungal isolate was identified to Phoma chrysanthemicola. Gram-negative bacteria were isolated from three plants out of 25 tested. They were identified using classical methods, the BIOLOG identification system based on the utilisation of 95 different carbon sources, and the MIDI system for the analysis of whole cell fatty acids. The two latter systems were computer-associated and utilised an extensive reference library of isolates. One bacterial isolate was identified as Enterobacter agglomerans and two isolates as Pseudomonas marginalis. R. solani AG-5-Gal reduced the emergence of Lupinus luteus, L. polyphyllus and french bean (Phaseolus vulgaris) and the growth of broad bean (Viciafaba), L. luteus and french bean, but did not cause obvious damage in goat's rue and pea (Pisum sativum). However, R. solani AG-5-Gal was re-isolated from the roots of all the test plant species following inoculation. P. chrysanthemicola reduced the emergence of L. polyphyllus and the growth of goat's rue, french bean and broad bean, and it was re-isolated from all of the test plant species (except for french bean) following inoculation. All the bacteria reduced the emergence of french bean, but not that of goat's rue and pea, when applied to the soil. When the roots were dipped into bacterial suspension, all the bacteria damaged french bean and L. polyphyllus. Additionally, P. marginalis JV3 damaged goat's rue and red clover. The pathogenicity of the fungi and bacteria were not changed when they were double-inoculated in pairs, except for R. solani AG-5-Gal and P. marginalis JV2 which reduced the emergence of goat's rue when inoculated together but not when inoculated separately.     

Influence of the mycorrhizal fungus,Glomus coronatum,and soil phosphorus on infection and disease caused by binucleate Rhizoctonia and Rhizoctonia solani on mung bean (Vigna radiata)

Root-infecting fungal pathogens and also parasites, which do not cause major disease symptoms cause problems of contamination in pot cultures of arbuscular mycorrhizal (AM) fungi. We investigated the effect of the AM fungus, Glomus coronatum Giovannetti on disease caused by binucleate Rhizoctonia sp. (BNR) and R. solani in mung bean in the absence (P0) and presence (P1) of added soil phosphorus (P). When G. coronatum and BNR or R. solani were inoculated at the same time, G. coronatum improved the growth of the plants and reduced colonization of roots by BNR, but not by R. solani. R. solani reduced the growth of non-mycorrhizal mung bean in P0 soil 6 weeks after inoculation, whereas BNR had no effect on growth. G. coronatum reduced the severity of disease caused by BNR or R. solani on mung bean in both soil P treatments. When G. coronatum was established in the roots 3 weeks before BNR or R. solani was added to the potting mix, there was no significant effect of BNR or R. solani on growth of mung bean. Prior colonization by G. coronatum slightly reduced indices of disease caused by BNR or R. solani. In both experiments, addition of P stimulated plant growth and reduced the colonization of roots by BNR, but had little effect on disease severity. We conclude that the reduction of the effect of BNR or R. solani on mung bean could not be explained by improved P nutrition, but could be attributed to the presence of G. coronatum within and among the roots.     

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.     

Root rot disease incidence and severity on some legume species grown in Samsun and the fungi isolated from roots and soils

Abstract

Root rot disease is very common in the bean, soybean, faba bean and pea plants growing areas in Samsun province. Disease incidence and severity were detected the highest at 93.8% and 55.4% in the bean growing area, and the lowest at 64.0% and 24.3% in the faba bean growing area respectively. In this study, a total of 2714 fungal isolates were obtained from some legume plants and soil samples. The most common fungi isolated from root and soil samples were Fusarium spp., multinucleate Rhizoctonia (MNR), binucleate Rhizoctonia (BNR) and Pythium spp. respectively. Fusarium spp. were isolated at high rates from all the examined areas. MN Rhizoctonia and BN Rhizoctonia were isolated both from inner and coastal areas of the province, whereas Pythium spp. were isolated in costal areas, except for the Vezirköprü district which is situated in the inner area. When looking at the interactions among pathogens causing root rot, it was found the great majority of the samples (30.4%) isolated both Fusarium spp. and MNR-BNR group fungi, whereas Fusarium spp. and Pythium spp. were isolated together from 10.9% of the samples and MNR-BNR and Pythium spp. from only 1.5% of the samples.     

Integrated control of root rot and wilt disease of faba bean by soil amendment with suppressive compost in combination with seed coating with an antagonistic yeast

For this study, 21 isolates of fungi belonging to Rhizoctonia and Fusarium genera were isolated from the diseased faba bean plants, obtained from the different localities in Assiut governorate, showing root rot and wilt symptoms. The isolates proved to be pathogenic on Masr 1 faba bean cultivar under greenhouse conditions. F. oxysporum isolates caused wilt disease; however, the isolates of R. solani and other Fusarium species caused root rot. The virulence of isolates on the tested faba bean cultivar was different. The highly pathogenic isolates of these fungi were employed in this study. The effect of soil amendment with Planta Rich and Rich Composts (CMs) alone or in combination with seed coating by the antagonistic yeast Pichia guilliermondi before sowing on the severity of Rhizoctonia and Fusarium root rot and Fusarium wilt of faba bean was tested under greenhouse and field conditions. The tested isolates of yeast proved to be highly antagonistic to the pathogen in vitro. The test rates of CMs were equivalent to 2, 7, 10 and 14 ton/feddan in the greenhouse and 7 and 10 t/feddan in the field conditions. Uncomposted soil was used as a control. The results showed that the tested CMs have a suppressive effect on the severity of root rot and wilt diseases of faba bean under greenhouse and field conditions. The application of CMs (Planta Rich and Rich) alone at the rates equivalent to 2, 7, 10 and 14 t/feddan in greenhouse and 7 and 10 t/feddan in the field conditions to the soil infested with the tested pathogens reduced percentage of the tested diseases compared with uncomposted soil. Combined CMs treatments with yeast seed treatment increased the suppressive effect of CMs on the disease severity.     

Chemotaxonomy of fungi in the <Emphasis Type="Italic">Rhizoctonia solani</Emphasis> species complex performing GC/MS metabolite profiling

Hyphal anastomosis testing and molecular methods have been the primary criteria employed to understand the evolutionary and taxonomic relationships of the soil-borne fungal plant pathogen Rhizoctonia solani species complex. In this study, a metabolomics-based approach for characterizing and identifying isolates of R. solani using gas chromatography/mass spectrometry (GC/MS) metabolite profiling and footprinting was developed. Multivariate and hierarchical cluster analyses of GC/MS data provided resolution of isolates belonging to anastomosis groups (AGs) 1–6, 9, and 10 of R. solani. Clustering of R. solani AG-3 isolates, based on host origin, was also observed and attributed to metabolite-biomarkers belonging to amino, carboxylic and fatty acids. The chemotaxonomic approach using metabolomics is a high-throughput methodology that complements existing molecular approaches for the taxonomic investigation of Rhizoctonia isolates and monitoring of fungal metabolism.     

Evolutionary Diversification Indicated by Compensatory Base Changes in ITS2 Secondary Structures in a Complex Fungal Species, <Emphasis Type="Italic">Rhizoctonia solani</Emphasis>

The rRNA cistron (18S–ITS1–5.8S–ITS2–28S) is used widely for phylogenetic analyses. Recent studies show that compensatory base changes (CBC) in the secondary structure of ITS2 correlate with genetic incompatibility between organisms. Rhizoctonia solani consists of genetically incompatible strain groups (anastomosis groups, AG) distinguished by lack of anastomosis between hyphae of strains. Phylogenetic analysis of internal transcribed spacer (ITS) sequences shows a strong correlation with AG determination. In this study, ITS sequences were reannotated according to the flanking 5.8S and 28S regions which interact during ribogenesis. One or two CBCs were detected between the ITS2 secondary structure of AG-3 potato strains as compared to AG-3 tobacco strains, and between these two strains and all other AGs. When a binucleate Rhizoctonia species related to Ceratobasidiaceae was compared to the AGs of R. solani, which were multinucleate (3–21 nuclei per cell), 1–3 CBCs were detected. The CBCs in potato strains of AG-3 distinguish them from AG-3 tobacco strains and other AGs yielding further evidence that the potato strains of AG-3 originally described as R. solani are a species distinct from other AGs. The ITS1–5.8S–ITS2 sequences were analyzed by direct sequencing of PCR products from 497 strains of AG-3 isolated from potato. The same 10 and 4 positions in ITS1 and ITS2, respectively, contained variability in 425 strains (86%). Nine different unambiguous ITS sequences (haplotypes) could be detected in a single strain by sequencing cloned PCR products indicating that concerted evolution had not homogenized the rRNA cistrons in many AG-3 strains. Importantly, the sequence variability did not affect the secondary structure of ITS2 and CBCs in AG-3. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.