Genetic diversity of Rhizoctonia solani AG-3 from potato and tobacco in North Carolina

Anastomosis group 3 (AG-3) of Rhizoctonia solani (teleomorph = Thanatephorus cucumeris) is frequently associated with diseases of potato (AG-3 PT) and tobacco (AG-3 TB). Although isolates of R. solani AG-3 from these two Solanaceous hosts are somatically related based on anastomosis reaction and taxonomically related based on fatty acid, isozyme and DNA characters, considerable differences are evident in their biology, ecology, and epidemiology. However, genetic diversity among field populations of R. solani AG-3 PT and TB has not been documented. In this study, the genetic diversity of field populations of R. solani AG-3 PT and AG-3 TB in North Carolina was examined using somatic compatibility and amplified fragment length polymorphism (AFLP) criteria. A sample of 32 isolates from potato and 36 isolates from tobacco were paired in all possible combinations on PDA plus activated charcoal and examined for their resulting somatic interactions. Twenty-eight and eight distinct somatic compatibility groups (SCG) were identified in the AG-3 PT and AG-3 TB samples, respectively. AFLP analyses indicated that each of the 32 AG-3 PT isolates had a distinct AFLP phenotype, whereas 28 AFLP phenotypes were found among the 36 isolates of AG-3 TB. None of the AG-3 PT isolates were somatically compatible or shared a common AFLP phenotype with any AG-3 TB isolate. Clones (i.e., cases where two or more isolates were somatically compatible and shared the same AFLP phenotype) were identified only in the AG-3 TB population. Four clones from tobacco represented 22% of the total population. All eight SCG from tobacco were associated with more than one AFLP phenotype. Compatible somatic interactions between AG-3 PT isolates occurred only between certain isolates from the same field (two isolates in each of four different fields), and when this occurred AFLP phenotypes were similar but not identical.     

Control of Fusarium solani rot of potato tubers with fungicides

Tecnazene (up to 33 ppm) and dichloran (up to 500 ppm) had little effect on germination of spores or growth of Fusarium solani isolated from and causing a rot of potato tubers; they also did not decrease rotting when applied to wounds later inoculated with the pathogen. Benomyl and thiabendazole (up to 500 ppm) also had little effect on spore germination but did greatly decrease growth at 5 ppm. A pronounced pink coloration developed in cultures growing slowly in the presence of benomyl; a similar though less striking effect appeared in agar cultures containing thiabendazole. Benomyl suspended in water or diluted with Fuller's earth gave good control of rotting when applied to wounds inoculated later with F. solani. Still better control was obtained with thiabendazole; dusts containing 1% a.i. substantially decreased rots and those containing 10 % a.i. gave almost complete control when applied to wounds shortly before inoculation. Thiabendazole was also very effective when used 24 h after inoculation and a fair measure of control was obtained when it was applied 24 h later. Benomyl and thiabendazole placed on apparently intact surfaces of tubers caused tissue 5 mm deep to become toxic to F. solani 10 days later, and, unexpectedly, this tissue prevented spore germination. Fuller's earth alone substantially decreased rotting. The results obtained suggest that dusts containing thiabendazole have some promise for the control of Fusarium rots of potato tubers, especially of early crops.     

Genetic structure of populations of Rhizoctonia solani AG-3 on potato in eastern North Carolina

A polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method was developed to identify and differentiate genotypes of Rhizoctonia solani anastomosis group 3 subgroup PT (AG-3 PT), a fungal pathogen of potato. Polymorphic co-dominant single-locus PCR-RFLP markers were identified after sequencing of clones from a genomic library and digestion with restriction enzymes. Multilocus genotypes were determined by a combination of PCR product and digestion with a specific restriction enzyme for each of seven loci. A sample of 104 isolates from one commercial field in each of five counties in eastern North Carolina was analyzed, and evidence for high levels of gene flow between populations was revealed. When data were clone-corrected and samples pooled into one single North Carolina population, random associations of alleles were found for all loci or pairs of loci, indicating random mating. However, when all genotypes were analyzed, the observed genotypic diversity deviated from panmixia and alleles within and between loci were not randomly associated. These findings support a model of population structure for R. solani AG-3 PT on potato that includes both recombination and clonality.     

Soil suppressiveness to Rhizoctonia solani and microbial diversity

Rhizoctonia solani anastomosis group 2-2IIIB causes damping-off, black root rot and crown rot in sugar beet (Beta vulgaris). Based on experiences of growers and field experiments, soils can become suppressive to R. solani. The fungus may be present in the soil, but the plant does not show symptoms. Understanding the mechanisms causing soil suppressiveness to R. solani is essential for the development of environmentally friendly control strategies of rhizoctonia root rot in sugar beet. A bioassay that discriminates soils in their level of disease suppressiveness was developed. Results of bioassays were in accordance with field observations. Preliminary results indicate an active role of microbial communities. Our research is focused on the disentanglement of biological mechanisms causing soil suppressiveness to R. solani in sugar beet. Therefore, we are handling a multidisciplinary approach through experimental fields, bioassays, several in vitro techniques and molecular techniques (PCR-DGGE).     

立枯丝核菌遗传多样性研究方法介绍

立枯丝核菌（Rhizoctonia solani Kühn）是一个集合种,遗传多样性丰富.关于遗传多样性的研究一直是R. solani研究的热点.本文就用于R. solani遗传多样性研究的方法进行了综述.分别解释并阐述了各种方法的优缺点,其中菌丝融合法是研究R. solani遗传多样性的传统方法,该法需借助显微镜且耗时耗力;同工酶法简便、高效、低廉,但常需要与其它方法联用;脂肪酸法操作难度小,价格相对较低,但该法受菌株生长状况和脂肪酸脂化方法的限制;分子标记法方法众多,各有利弊,通过比较发现rDNA-ITS是研究R. solani遗传多样性比较合适的方法.本文还介绍了不同方法在R. solani遗传多样性研究中的具体应用.     

Genetic manipulation of 6-phosphofructokinase in potato tubers

The aim of this work was to discover whether genetic manipulation of 6-phosphofructokinase [EC 2.7.1.11; PFK(ATP)] influenced the rate of respiration of tuber tissue of Solanum tuberosum L. Transgenic plants were produced that contained the coding sequence of the Escherichia coli pfkA gene linked to a patatin promoter. Expression of this chimaeric gene in tubers resulted in a 14to 21-fold increase in the maximum catalytic activity of PFK(ATP) without affecting the activities of the other glycolytic enzymes. Tubers, and aged disks of tuber tissue, from transformed plants showed no more than a 30% fall in the content of hexose 6-monophosphates; the other intermediates of glycolysis increased threeto eightfold. Fructose-2,6-bisphosphate was barely detectable in aged disks of transformed tubers. The relative rates of ¹⁴CO₂ production from [1-¹⁴C]-and [6-¹⁴C]-glucose supplied to disks of transformed and control tubers were similar. Oxygen uptake and CO₂ production by aged disks of transformed tubers did not differ significantly from those from control tubers. The same was true of CO₂ production, in air, and in nitrogen, for tuber tissue. It is concluded that PFK(ATP) does not dominate the control of respiration in potato tubers.Abbreviations Fru2,6bisP fructose-2,6-bisphosphate - FW freshweight - GUS -glucuronidase - PFK(ATP) 6-phosphofructokinase - PFK(PPi) pyrophosphate: fructose-6-phosphate 1-phosphotransferase     

中国北方马铃薯黑痣病立枯丝核菌的融合群鉴定

从山东、甘肃、青海、内蒙古、河北和黑龙江6省采集马铃薯黑痣病标本300余份,分离获得251个立枯丝核菌Rhizoctonia solani菌株。融合群测定结果表明,这些菌株分别属于多核的丝核菌AG‐3、AG1‐IB、AG4‐HG‐Ⅰ、AG4‐HG‐Ⅱ、AG4‐HG‐Ⅲ、AG‐5和AG‐11融合群。其中AG‐3是优势致病群,占分离菌株总数的71.31%;其次是AG4‐HG‐Ⅰ,占15.14%;AG‐11融合群菌株是国内首次从罹病马铃薯植株上分离得到。从各融合群中选取代表性的菌株进行5.8S rDNA‐ITS区序列分析,结果表明,隶属不同融合群或亚群菌株的5.8S rDNA‐ITS区序列存在较大的差异,而相同融合群(亚群)不同菌株的序列具有较高一致性。     

山东省玉米纹枯菌融合群类型及遗传多样性

从山东省14个县市区采集的玉米纹枯病标本上分离获得103个玉米纹枯菌菌株。核荧光染色确定菌丝细胞核的数目,以及利用配对培养法确定不同菌株细胞是否融合。结果表明这些菌株分别属于多核丝核菌的AG-1-IA、AG-1-IB、AG-1-IC、AG-3、AG-4-HG-I、AG-5和WAG-Z融合群和双核丝核菌的AG-Ba融合群,其中AG-1-IA类型菌株数量占菌株总数的60.19%,为优势融合群。通过inter-simple sequence repeats（ISSR）标记技术进行菌株的遗传多样性分析,获得45个ISSR分子标记,其中91.1%的片段具有多态性,表明种群间存在丰富的遗传多样性。UPGMA聚类分析将103个菌株分成6个遗传聚类群,遗传聚类群的菌株组成说明遗传群组的划分与菌株的地理来源和菌株融合群类型均存在一定的相关性。     

Stem canker (Rhizoctonia solani) on five early and seven maincrop potato cultivars I. Infection of shoots, stolons and tubers

In 1983 and 1984, potato seed tubers of five early and seven maincrop cultivars were inoculated with cultures of Rhizoctonia soluni during planting to simulate severe seed infection. Shoot and stolon infection was assessed in June-August and black scurf on tubers recorded after harvest in October. Almost all shoots of all cultivars had stem canker in both years and disease on shoots, stolons and tubers was more severe in 1984 than in 1983. In 1983 similar amounts of disease developed on all early cultivars and between 11% (Ulster Sceptre) and 32% (Maris Peer) shoots were pruned off. Maris Peer had a stem canker score lower than other cultivars in 1984 but more than half the shoots were pruned off. Shoot pruning on Estima, Ulster Prince and Ulster Sceptre was more common on plants from sprouted than non-sprouted seed. Between 30 and 50% of stolons were pruned off. After harvest in 1985, black scurf was least prevalent on Arran Comet and Maris Peer tubers and in 1984 on Arran Comet and Estima tubers from non-sprouted seed. Of the maincrop cultivars, King Edward plants from sprouted seed had many shoots pruned off in both years. Shoot pruning was also prevalent on Maris Piper and Pentland Squire plants from non-sprouted seed. Record had fewest pruned shoots and stolons and the lowest stem canker score. The disease was more severe on Pentland Crown and Maris Piper plants from non-sprouted than sprouted seed. Black scurf was most common on Cara and King Edward tubers in 1983 and on King Edward and Record tubers in 1984. In both years few shoots but many tubers were infected on plants from non-inoculated seed and the significance of this is discussed.     

Luminescence-based detection of Erwinia carotovora associated with rotting potato tubers

A luminescence-based marker system has been used to follow rotting of potato tubers by Erwinia carotovora. The early stages of infection could be detected by visual observation of areas of luminescence developing from the point of inoculation. During later stages luminescence was limited to peripheral regions and to central regions where the structure of the potato was destroyed. Luminometry demonstrated reduction in aerobic activity of infecting cells due to oxygen limitation. The system enables rapid and sensitive detection of active marked populations and distinction between aerobic and fermentative activity.     

Diversity of Rhizoctonia solani associated with pulse crops in different agro-ecological regions of India

Four hundred seventy Rhizoctonia solani isolates from different leguminous hosts originating from 16 agro-ecological regions of India covering 21 states and 72 districts were collected. The disease incidence caused by R. solani varied from 6.8 to 22.2 % in the areas surveyed. Deccan plateau and central highlands, hot sub-humid ecoregion followed by northern plain and central highlands and hot semi-arid ecoregion showed the highest disease incidence. R. solani isolates were highly variable in growth diameter, number, size and pattern of sclerotia formation as well as hyphal width. The isolates obtained from aerial part of the infected plants showing web blight symptoms produced sclerotia of 1–2 mm in size whereas, the isolates obtained from infected root of the plants showing wet root rot symptoms produced microsclerotia (<1 mm). Majority of R. solani isolates showed <8 μm hyphal diameter. Based on morphological characters the isolates were categorized into 49 groups. Seven anastomosis groups (AGs) were identified among the populations of R. solani associated with the pulse crops. The frequency (25.6 %) of AG3 was the highest followed by AG2–3 (20.9 %) and AG5 (17.4 %). The cropping sequence of rice/sorghum/wheat-chickpea/mungbean/urdbean/cowpea/ricebean influenced the dominance of AG1 (16.3 %). Phylogenetic analysis utilizing ITS-5.8S rDNA gene sequences indicated high level of genetic similarity among isolates representing different AGs, crops and regions. ITS groups did not correspond to the morphological characters. The sequence data from this article has been deposited with NCBI data libraries with JF701707 to JF701795 accession numbers.     

Changes in the biochemical composition of stored potato tubers during pathogenesis by Rhizoctonia bataticola

The nutrient contents of the two cultivare of Potato‐Irish Cobbler and Red Pontiac were quantitatively altered by Rhizoctonia bataticola during pathogenesis. The glucose content of both cultivare increased whereas sucrose, maltose and fructose contents decreased appreciably in quantity during the period of incubation. The total protein and lipid contents of both cultivare were also lowered quantitatively by the rot‐causing organism. The depletion of total carbohydrate, protein and lipid contents, was more in Irish Cobbler cultivar than in Red Pontiac cultivar.     

Molecular characterisation of an endornavirus from Rhizoctonia solani AG-3PT infecting potato

Rhizoctonia solani (teleomorph: Thanatephorus cucumeris) is a soil-borne plant pathogenic fungus that has a broad host range, including potato. In this study, the double-stranded RNA (dsRNA) profiles were defined for 39 Rhizoctonia solani isolates representative of two different anastomosis groups (AGs) associated with black scurf of potato in New Zealand. A large dsRNA of c. 12 kb–18 kb was detected in each of the isolates, regardless of AG or virulence on potato. Characterisation of the large dsRNA from R. solani AG-3PT isolate RS002, using random amplification of total dsRNA and analyses of overlapping cDNA sequences, resulted in the assembly of a consensus sequence of 14 694 nt. A single, large open reading frame was identified on the positive strand of the assembled sequence encoding a putative polypeptide of at least 4893 amino acids, with a predicted molecular mass of 555.6 kDa. Conserved domains within this polypeptide included those for a viral methyltransferase, a viral RNA helicase 1 and an RNA-dependent RNA polymerase. The domains and their sequential organisation revealed the polyprotein was very similar to those encoded by dsRNA viruses of the genus Endornavirus, in the family Endornaviridae. This is the first report of an endornavirus in R. solani, and thus the putative virus is herein named Rhizoctonia solani endornavirus - RS002 (RsEV-RS002). Partial characterisation of the large dsRNAs in five additional AG-3PT isolates of R. solani also identified them as probable endornaviruses, suggesting this family of viruses is widespread in R. solani infecting potato. The ubiquitous nature of endornaviruses in this plant pathogen implies they may have an important, but yet uncharacterised, role in R. solani.     

The properties of l-fucose binding agglutinin associated with the cell wall of Rhizoctonia solani

The adherence of Escherichia coli B cells to cell wall associated-agglutinin of the soil borne plant pathogen Rhizoctonia solani, was inhibited by l-fucose, l-galactose, trypsin, SDS, cycloheximide and Na₂-EDTA. The coiling of the biocontrol agent Trichoderma harzianum around Rhizoctonia hyphae was prevented by SDS, cycloheximide, Na₂-EDTA and methyl--l-fucoside — an inhibitor of Rhizoctonia agglutinin not metabolized by both fungi. The possible role of the agglutinin in Trichoderma-Rhizoctonia interaction is discussed.     

Control of pre- and post-emergence damping-off of potato seedlings caused by Rhizoctonia solani in Peru

Fungicide treatment of soil and true potato seed and the use of subsoil as a growth medium significantly reduced pre-emergence damping-off caused by Rhizoctonia solani. Attempts were made to determine safe levels of fungicides for soil and seed treatments. For seed, safe levels varied between 0–9% a.i. for pentachloronitrobenzene (PCNB) and 3-0% a.i. for mancozeb (dithane M-45). Fungicides only marginally reduced post-emergence damping-off of transplants raised from true potato seed but gave significant increase in yield. The prospects of using fungicide treatment of true seed to control damping-off in seed beds and in direct field sowing are discussed.     

Methylation and aging in Rhizoctonia solani

Our earlier studies had shown that as fungi age, many of their vital functions decrease; in Rhizoctonia solani, protein synthesis is one of the functions so affected. We now find that the ability to methylate tRNA, a vital component of the protein synthesizing system, also decreases with age. This methylation of Escherichia coli tRNA by R. solani methylase preparations increased with the concentration of enzyme and with time of incubation; in both cases the rate of increase was considerably higher for preparations from young cells than for those from old cells. The methylation reaction also increased with the concentration of substrate tRNA, with temperature, at least to 45° C, and with pH to 9.0. Methylase preparations from R. solani methylated both exogenous E. coli tRNA and yeast tRNA, but were only weakly active on isolated R. solani tRNA. However, acid-precipitated methylases from R. solani were very effective in methylating the homologous exogenous tRNA. Regardless of the source of the tRNA used as substrate, the methylases from older cells were always less active than those from young cells from the same mycelium. No methylase inhibitor was detected in the fungus.     

Genetic Variability in Pectic Enzymes of Rhizoctonia solani Isolates Causing Bare-patch Disease of Cereals

Field isolates of Rhizoctonia solani obtained from three discrete bare patches in a wheat field in Western Australia were characterized by pectic zymogram grouping. The genetic background of pectic enzymes was analysed by comparing the zymograms of asexual homokaryons and sexual progenies derived from field isolates. The 170 field isolates obtained from the field site produced indistinguishable pectic zymograms. However, variations among field isolates of the same zymogram group were detected, on the basis of zymograms of their resultant protoplast-regenerated cultures. Asexual sibling homokaryons derived from each of the field isolates were heterogeneous for their pectic enzymes. Homokaryons with a common heterokary on incompatibility factor, obtained from a field isolates were homogeneous for pectic enzymes. Basidiospore progenies of a field isolate segregated widely in pectic zymograms. It appeared that the expression of pectic enzymes by field isolates involved multiple genetic factors. The variation of zymograms among homokaryotic strains suggests that each field isolate of R. solani contains two types of nuclei, although cells of vegetative hyphae are multinucleate.     

Characterization of Rhizoctonia solani associated with Sore Shin and Leaf Spot Symptoms on Tobacco in Zimbabwe

The relationship between Rhizoctonia solani isolates derived from leaf spot and sore shin symptoms on tobacco was characterized by determining nuclear number, hyphal diameter, anastomosis grouping (AG) and pathogenicity. The leaf spot isolates had significantly more nuclei per cell, and wider hyphae than the sore shin isolates. Anastomosis pairings with known tester strains confirmed the leaf spot isolates to be AG 3, and the sore shin isolates to be AG 4. Pathogenicity testing indicated that the leaf spot isolates were pathogenic to all parts of the plant, whereas the sore shin isolates tended to form lesions on stems only.