Characterization of Rhizoctonia solani AG-3 Isolates Causing Target Spot of Flue-Cured Tobacco in China

2013 ◽  
Vol 726-731 ◽  
pp. 4321-4325 ◽  
Author(s):  
Yan Qin Zhao ◽  
Yuan Hua Wu ◽  
Ying Fu ◽  
Meng Nan An ◽  
Jian Guang Chen ◽  
...  

Rhizoctonia solani Kühn is the causal pathogen of tobacco target spot, a serious fungal disease of tobacco that severely impairs yield and quality in northeast China. The objective of this study was to characterize isolates ofR. solanifrom tobacco in China. Among 58Rhizoctoniaisolates examined, all of them were multinucleate. Phylogenetic analyses and hyphal anastomosis criteria suggest that the isolates belonged toR. solanianastomosis group (AG) 3. Target spot isolates from Liaoning province formed a single phylogenetic group together with tomato isolates ofR. solaniAG-3 from Japan and are more closely related toR. solaniAG-3 isolates in tomato and potato than that in tobacco from USA. Pathogenicity test for each isolates was fulfilled using a method of inoculating tobacco leaves from plants grown for 8 weeks (cv. NC89).

2014 ◽  
Vol 1010-1012 ◽  
pp. 1161-1164
Author(s):  
Yan Qin Zhao ◽  
Yuan Hua Wu ◽  
Xiu Xiang Zhao ◽  
Meng Nan An ◽  
Jian Guang Chen ◽  
...  

Rhizoctonia solaniKühn is a causal pathogen responsible for many types of plant disease worldwide and a major soilborne fungal pathogen that severely impairs yield and quality of tobacco worldwide. Activities, pathogenicity of the cell wall-degrading enzymes produced by theRhizoctoniasolanifrom tobacco target spot disease both in liquid medium and in tobacco tissue were studied. The result showed thatR.solanifrom tobacco can produce pectinase and cellulase both in vitro and vivo, and the activity of PG and PMG was the highest in vitro. The activity of Cx and β-glucosidase was the highest in vivo, and enzyme production ability of strong pathogenicity strains is stronger than the weak pathogenicity strains in vitro.


2014 ◽  
Vol 1010-1012 ◽  
pp. 80-83 ◽  
Author(s):  
Yan Qin Zhao ◽  
Yuan Hua Wu ◽  
Xiu Xiang Zhao ◽  
Meng Nan An ◽  
Jian Guang Chen

Rhizoctonia solani Kühn is the causal pathogen of tobacco target spot, a major soilborne fungal pathogen that severely impairs yield and quality of tobacco worldwide. It is difficult to identify and quality the pathogen in plant and soil using conventional methods. In the study we developed a real-time, quantification polymerase chain reaction (QPCR) assay to detect and quantify R. solani AG-3 DNA from infected tobacco tissue and soil. Using a specific primer pair based on the internal transcribed spacer region of the fungal DNA sequence, R. solani AG-3 DNA at quantities as low as 100 fg of purified pathogen DNA could be successfully detect.


2020 ◽  
Vol 86 (6) ◽  
pp. 457-467
Author(s):  
Tomoo Misawa ◽  
Daisuke Kurose ◽  
Kuniaki Shishido ◽  
Takeshi Toda ◽  
Shiro Kuninaga

Plant Disease ◽  
2012 ◽  
Vol 96 (3) ◽  
pp. 456-456 ◽  
Author(s):  
G. Mercado Cárdenas ◽  
M. Galván ◽  
V. Barrera ◽  
M. Carmona

In August 2010, lesions similar to those reported for target spot were observed on Nicotiana tabacum L. plants produced in float systems in Cerrillos, Salta, Argentina. Tobacco leaves with characteristic lesions were collected from different locations in Cerrillos, Salta. Symptoms ranged from small (2 to 3 mm), water-soaked spots to larger (2 to 3 cm), necrotic lesions that had a pattern of concentric rings, tears in the centers, and margins that often resulted in a shot-hole appearance. Isolation of the causal agent was made on potato dextrose agar (PDA) acidified to pH 5 with 10% lactic acid and incubated at 25 ± 2°C in darkness for 2 to 3 days. Hyphal tips were transferred to a new medium and the cultures were examined for morphological characters microscopically (3). Eight isolates were obtained. The rapid nuclear-staining procedure using acridine orange (3) was used to determine the number of nuclei in hyphal cells. Multinucleate hyphae were observed, with 4 to 9 nuclei per cell. Molecular characterization was conducted by examining the internal transcribed spacer (ITS) region from all of the isolates of the pathogen identified as Rhizoctonia solani based on morphological characteristics (1). Fragments amplified using primers ITS1 (5′TCCGTAGGTGAACCTGCGG3′) and ITS4 (5′TCCTCCGCTTATTGATATGC3′) (4) were sequenced and compared with R. solani anastomosis group (AG) sequences available in the NCBI GenBank database. Sequence comparison identified this new isolate as R. solani anastomosis group AG 2-1. Previous isolates of target spot were identified as AG 3 (2). The isolates that were studied were deposited in the “Laboratorio de Sanidad Vegetal” INTA-EEA-Salta Microbial Collection as Rs59c, Rs59b, Rs59, Rs66, Rs67, Rs68, Rs69, and Rs70. The ITS nucleotide sequence of isolate Rs59 has been assigned the GenBank Accession No. JF792354. Pathogenicity tests for each isolate were performed using tobacco plants grown for 8 weeks at 25 ± 2°C with a 12-h photoperiod. Ten plants were inoculated by depositing PDA plugs (0.2 cm) colonized with R. solani onto leaves; plants inoculated with the pure PDA plug without pathogen served as controls. The plants were placed in a 25 ± 2°C growth chamber and misted and covered with polyethylene bags that were removed after 2 days when plants were moved to a glasshouse. After 48 h, symptoms began as small (1 to 2 mm), circular, water-soaked spots, lesions enlarged rapidly, and often developed a pattern of concentric rings of 1 to 2 cm. After 8 days, all inoculated plants showed typical disease symptoms. Morphological characteristics of the pathogen reisolated from symptomatic plants were consistent with R. solani. Control plants remained healthy. These results correspond to the first reports of the disease in the country. Compared to other areas in the world, target spot symptoms were only observed in tobacco plants produced in float systems and were not observed in the field. The prevalence of the disease in Salta, Argentina was 7%. To our knowledge, this is the first report of R. solani AG2.1 causing target spot of tobacco. References: (1) M. Sharon et al. Mycoscience 49:93, 2008. (2) H. Shew and T. Melton. Plant Dis. 79:6, 1995. (3) B. Sneh et al. Identification of Rhizoctonia species. The American Phytopathological Society, St. Paul, MN, 1991. (4) T. J. White et al. Page 282 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, 1990.


2001 ◽  
Vol 91 (11) ◽  
pp. 1054-1061 ◽  
Author(s):  
Achmadi Priyatmojo ◽  
Verma E. Escopalao ◽  
Naomi G. Tangonan ◽  
Cecilia B. Pascual ◽  
Haruhisa Suga ◽  
...  

A new foliar disease on coffee leaves was observed in Mindanao, Philippines, in 1996. The symptoms appeared as large circular or irregularly shaped necrotic areas with small circular necrotic spots (1 mm or less in diameter) usually found around the periphery of the large necrotic areas. Rhizoctonia solani was consistently isolated from these diseased coffee leaves. Isolates obtained were multinucleate (3 to 12 nuclei per hyphal cell), had an optimum temperature for hyphal growth at 25°C, prototrophic for thiamine, and anastomosed with tester isolates belonging to R. solani anastomosis group 1 (AG-1). Mature cultures on potato dextrose agar (PDA) were light to dark brown. Sclerotia, light brown to brown, were formed on the surface of PDA and covered the whole mature colony culture. Individual sclerotia often aggregated into large clumps (3 to 8 mm in diameter) and their color was brown to dark brown. In pathogenicity tests, isolates from coffee caused necrotic symptoms on coffee leaves, whereas isolates of AG-1-IA (not isolated from coffee), 1-IB, and 1-IC did not. The results of analyses of restriction fragment length polymorphism of ribosomal DNA internal transcribed spacer, random amplified polymorphism DNA, and fatty acid profiles showed that R. solani isolates from coffee are a population of AG-1 different from AG-1-IA, 1-IB, and 1-IC. These results suggest that R. solani isolates from coffee represent a new subgroup distinct from AG-1-IA, 1-IB, and 1-IC. A new subgroup ID (AG-1-ID) is proposed.


Plant Disease ◽  
2013 ◽  
Vol 97 (8) ◽  
pp. 1119-1119 ◽  
Author(s):  
A. Garibaldi ◽  
D. Bertetti ◽  
P. Pensa ◽  
A. Poli ◽  
M. L. Gullino

Origanum vulgare L., common name oregano, family Labiatae, is grown for its aromatic and medicinal properties and as ornamental. In the fall of 2012, a blight was observed in a farm located near Albenga (northern Italy) on 6% of 30,000 50-day-old plants, grown in trays in a peat/perlite mix. Semicircular, water soaked lesions appeared on leaves and stems, starting from the basal ones. As the disease progressed, blighted leaves turned brown, withered, clung to the shoots, and matted on the surrounding foliage. Eventually, infected plants died. Leaf and stem fragments taken from the margin of the diseased tissues belonging to 10 plants were disinfected for 10 s in 1% NaOCl, rinsed with sterile water, and plated on potato dextrose agar (PDA). A fungus with the morphological characters of Rhizoctonia solani was consistently recovered. Three isolates of R. solani obtained from affected plants were successfully anastomosed with R. solani isolate AG 1 (ATCC 58946). Three pairings were made for each tester strain. The hyphal diameter at the point of anastomosis was reduced, the anastomosis point was obvious, and death of adjacent cells was observed. Results were consistent with other reports on anastomosis reactions (2). Isolates from oregano were paired with R. solani isolates AG 2, 3, 4, 6, 7, or 11 and examined microscopically. Anastomosis was not observed in any of the pairings. Tests were conducted twice. Mycelium of 10-day-old isolates from oregano appeared reddish brown, coarse, and radiate. Numerous dark brown sclerotia, 0.3 to 1.0 mm diameter (average 0.7) developed within 10 days after transfer of mycelia to PDA in 90 mm diameter petri dishes at 21 to 24°C. The descriptions of mycelium and sclerotia were typical for subgroup IB Type 1 (4). The internal transcribed spacer (ITS) region of rDNA was amplified using the primers ITS1/ITS4 and sequenced. BLASTn analysis (1) of the 538 bp showed a 99% homology with the sequence of R. solani FJ746937, confirming the morphological identification of the species. The nucleotide sequence has been assigned the GenBank Accession KC493638. For pathogenicity tests, one of the isolates assigned to the anastomosis group AG-1-IB was tested by placing 9 mm diameter mycelial disks removed from PDA 10-day-old cultures of the fungus on leaves of 90-day-old oregano plants (n = 35). Thirty-five plants inoculated with non-inoculated PDA disks served as controls. Plants were covered with plastic bags and maintained in a growth chamber at 25 ± 1°C with 12 h light/dark. The first symptoms, similar to those observed in the farm, developed 3 days after inoculation. Nine days after the artificial inoculation, 50% of plants were dead. About 10 colonies of R. solani were reisolated from infected leaves of inoculated plants. Control plants remained healthy. The pathogenicity test was carried out twice with similar results. Symptoms caused by R. solani have been recently observed on O. vulgare in Greece (3). This is, to our knowledge, the first report of blight of O. vulgare caused by R. solani in Italy. References: (1) S. F. Altschul et al. Nucleic Acids Res., 25:3389, 1997. (2) D. E. Carling. Grouping in Rhizoctonia solani by hyphal anastomosis reactions. In: Rhizoctonia Species: Taxonomy, Molecular Biology, Ecology, Pathology and Disease control. Kluwer Academic Publishers, The Netherlands, pp. 37-47, 1996. (3) C. D. Holevas et al. Benaki Phytopathol. Inst., Kiphissia, Athens, 19:1-96, 2000. (4) R. T. Sherwood. Phytopathology 59:1924, 1969.


Plant Disease ◽  
2012 ◽  
Vol 96 (2) ◽  
pp. 288-288 ◽  
Author(s):  
X. Liao ◽  
Y. Fu ◽  
S. Zhang ◽  
Y. P. Duan

Indian spinach (Basella rubra L.) is a red stem species of Basella that is cultivated worldwide as an ornamental and the aerial parts are also consumed as a vegetable. In May of 2011, symptoms of damping-off were observed on approximately 10% of the plants at the stem base around the soil line of seedlings in a greenhouse in Homestead, FL. Lesions were initially water soaked, grayish to dark brown, irregular in shape, and sunken in appearance on large plants, causing the infected seedlings to collapse and eventually die. Symptomatic stem tissue was surface sterilized with 0.6% sodium hypochlorite, rinsed in sterile distilled water, air dried, and plated on potato dextrose agar (PDA). Plates were incubated at 25°C in darkness for 3 to 5 days. A fungus was isolated in all six isolations from symptomatic tissues on PDA. Fungal colonies on PDA were light gray to brown with abundant growth of mycelia, and the hyphae tended to branch at right angles when examined under a microscope. A septum was always present in the branch of hyphae near the originating point and a slight constriction at the branch was observed. Neither conidia nor conidiophores were found from the cultures on PDA. The characteristics of hyphae, especially the right angle branching of mycelia, indicate close similarity to those of Rhizoctonia solani (2,3). The internal transcribed spacer (ITS) region of rDNA was amplified with the primers ITS1/ITS4 and sequenced (GenBank Accession No. JN545836). Subsequent database searches by the BLASTN program indicated that the resulting sequence had a 100% identity over 472 bp with the corresponding gene sequence of R. solani anastomosis group (AG) 4 (GenBank Accession No. JF701752.1), a fungal pathogen reported to cause damping-off on many crops. Pathogenicity was confirmed through inoculation of healthy India spinach plants with the hyphae of isolates. Four 4-week-old plants were inoculated with the isolates by placing a 5-mm PDA plug of mycelia at the stem base and covering with a thin layer of the soil. Another four plants treated with sterile PDA served as a control. After inoculation, the plants were covered with plastic bags for 24 h and maintained in a greenhouse with ambient conditions. Four days after inoculation, water-soaked, brown lesions, identical to the symptoms described above, were observed on the stem base of all inoculated plants, whereas no symptoms developed on the control plants. The fungus was isolated from affected stem samples, and the identity was confirmed by microscopic appearance of the hyphae and sequencing the ITS1/ITS4 intergenic spacer region, fulfilling Koch's postulates. This pathogenicity test was conducted twice. R. solani has been reported to cause damping-off of B. rubra in Ghana (1) and Malaysia (4). To our knowledge, this is the first report of damping-off caused by R. solani AG-4 on Indian spinach in Florida and the United States. With the increased interest in producing Asian vegetables for food and ornamental purposes, the occurrence of damping-off on Indian spinach needs to be taken into account when designing programs for disease management in Florida. References: (1) H. A. Dade. XXIX. Bull. Misc. Inform. 6:205, 1940. (2) J. R. Parmeter et al. Phytopathology 57:218, 1967. (3) B. Sneh et al. Identification of Rhizoctonia species. The American Phytopathological Society, St Paul, MN, 1991. (4) T. H. Williams and P. S. W. Liu. Phytopathol. Pap. 19:1, 1976.


1999 ◽  
Vol 89 (5) ◽  
pp. 414-420 ◽  
Author(s):  
S. Banniza ◽  
A. A. Sy ◽  
P. D. Bridge ◽  
S. A. Simons ◽  
M. Holderness

Isolates of Rhizoctonia solani were obtained from plant and soil samples that had been systematically collected in a field experiment in Côte d'Ivoire to study the diversity of the pathogen and the influence of three different rice rotations on the pathogen population. Characterization by morphology, anastomosis testing, pathogenicity testing, and restriction fragment length polymorphisms (RFLPs) of AT-rich DNA (AT-DNA) showed that there were no differences in isolates from different experimental plots, suggesting that the soil as well as the plant population of the fungus was indistinguishable throughout the experiment and was not influenced by crop rotation. Analysis of AT-DNA showed that the isolates obtained from plant material and one from soil shared a distinct banding pattern, identical with the AT-DNA RFLP obtained for the reference strain of anastomosis group 1 (AG-1). The remaining soil isolates produced a consistent RFLP pattern that was distinct from that of the plant isolates. Morphological characterization of isolates produced two major clusters consisting of the same groups of isolates as found by AT-DNA RFLP. Diversity in morphological characters was much higher in plant than in soil isolates and indicated that the population might consist of several clones. Anastomosis testing revealed that soil as well as plant isolates were able to fuse with the tester strain of AG-1. Significant differences in disease severity were observed between the two groups of isolates in pathogenicity tests on rice plants, with plant isolates being distinctively more virulent.


1993 ◽  
Vol 41 (2) ◽  
pp. 253 ◽  
Author(s):  
HA Yang ◽  
K Sivasithamparam ◽  
PA Obrien

Field isolates of Rhizoctonia solani anastomosis group (AG) 8, the most important causal pathogen of cereal bare-patch disease, were paired with each other and with tester strains of other AGs on potato-dextrose agar amended with charcoal (PDCA) to investigate mycelial interactions. Pairings among AG 8 field isolates produced compatible interactions of either tuft or merging reactions. Tufts formed between all paired field isolates from different pectic zymogram groups (ZGs) within AG 8, but pairings between genetically identical isolates showed merging reactions. Pairings of AG 8 field isolates with the tester strains of the other AGs led to incompatible interactions varying from merging line to barrage reactions. As formation of a tuft indicates that the paired isolates are able to anastomose and to form viable heterokaryons, the testing of mycelial interaction types, highlighted by tuft formation, may be used as a rapid procedure to characterise field isolates of R. solani obtained from cereals.


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