Anastomosis Reactions Research Articles

Characterization of three-nucleate Rhizoctonia AG-E based on their morphology and phylogeny

The genus Rhizoctonia has been classified into two main groups according to the number of nuclei. Binucleate Rhizoctonia strains have two nuclei in each cell, whereas multinucleate Rhizoctonia fungi were observed to have a variable number of nuclei ranging from 4 to 16 in each cell. In the study, twelve Polish isolates were tested. According to ITS1-5,8S-ITS2 rDNA sequences, the isolates were classified in the AG-E. Their affiliation to AG was confirmed by anastomosis reactions with tester isolates. The number of nuclei was counted with DAPI staining under a fluorescent microscope, and the diameter of the hyphae was also measured. Not all AG-E isolates had the same number of nuclei in their cells: one group among these fungi produced cells with a diverse number of nuclei, usually 3; however, this number ranged from 2 to 4, making the average number of nuclei close to 3. It can be assumed that all isolates with three nuclei belong to this group, which may greatly facilitate the preliminary identification of trinucleate isolates of Rhizoctonia spp. belonging to AG-E. Based on these characters, we call these isolates AG-E-3n isolates. The thiamine requirement is not helpful in classifying and describing the AG-E strains.

First Report of Target Spot Caused by Rhizoctonia solani AG-5 on Tobacco in China.

Tobacco is one of the vital economic crops in China. Nevertheless, tobacco diseases cause substantial economic losses each year. Tobacco target spot is a fungal disease which commonly found on the leaves. While both sexual and asexual reproduction can occur, asexual reproduction is much more common in tobacco. In June 2022, target spot was found on tobacco leaf samples from Yibin, Sichuan Province and Meitan, Guizhou Province, China. The typical symptoms were light brown tissue with concentric ring marks, and the necrotic part of the disease spot was fragile and forming perforation after falling off. The diseased tissue in the sample was cut off and sterilized in 75% ethanol for 1 min, and rinsed three times in sterilized distilled water. Finally, the tissues were placed on potato glucose agar (PDA) medium with kanamycin (0.1 mg/mL). After incubation at 28 °C in darkness for 3 days,the culture of the isolate grew in the form of radial mycelium on PDA dishes, the mycelium was white initially, turned brown generally at the later stage, and finally thickened and separated with the growth of the culture. Nine pathogenic strains were isolated, including four isolates from Yibin and five from Meitan. They were all used for pathogen identification. Genomic DNA of each isolate was extracted using the CATB method, and PCR analysis was performed with primers specifically designed to detect individual fusion groups or fusion subgroups of solani: AG-1 IA, IB, and IC; AG-3 PT; AG-4 HG-I, HG-II and HG-III; AGs-5-6 and P-21-22. Among the 11 specific primer pairs, only AG-5-specific primer amplified the fungal DNA, indicating that the nine isolates tested all belonged to the R. solani AG-5 fusion group. BLASTn search was performed on the gene sequences obtained from these strains and they deposited in GenBank under accession no. OP647851-OP647859. These gene sequences were aligned with the voucher specimen R. solani AG-5, with more than 99% similarity . The nine isolates were then tested for mycelial anastomosis reactions using the R. solani AG-5 standard strain following the method described by Ogoshi (1987). A decrease in the diameter of the mycelia at the anastomosis site and death of adjacent cells were observed, indicating their anastomosis response. Therefore, these nine strains were identified as R. solani AG-5 based on morphological and genetic analysis. Subsequently, one pathogenic strain from Meitan and another one from Yibin were selected for pathogenicity verification. Mycelial PDA blocks (6 mm in diameter) of the two isolates were inoculated on healthy tobacco plants, while leaves containing only PDA blocks were used as controls. A total of 6 replicates were conducted. After inoculation, they were incubated at 85% relative humidity and 15 to 25 °C. Koch's hypothesis was confirmed by reisolating pathogens from diseased leaves 5 days after inoculation. Typical symptoms were observed on tobacco plants inoculated with the pathogen strains but not on control tobacco plants. To the best of our knowledge, tobacco target spot has been reported caused by R. solani AG-3, AG-6 and AG-2.1 groups in the field in China and in Argentina. Up until now, this is the first report of R. solani AG-5 causing tobacco target spot on tobacco in the field in China. It was also found to be highly virulent to chickpea in Turkey. Due to serious damages caused by this disease in the last five years in China, more attention should be paid in disease control measures to avoid economic losses. In addition, it also provides some theoretical help for the damage caused by this pathogen on other hosts and helps people to better understand Rhizoctonia solani AG-5.

First Report of Target Spot Caused by Rhizoctonia solani AG-6 in Tobacco in China.

Tobacco is an annual and solanaceous crop, which is widely produced in China. In July 2020, tobacco target spot was observed on 50% of tobacco plants in a 5-ha commercial field of Bijie (27.32° N, 105.29° E), Guizhou province, China. Typical symptoms firstly appeared on the old leaves as round watery spots. Then the spots became a diameter of 2 to 20 cm, with concentric ring lines and dead spots. Fifteen small pieces (5 × 5 mm) of leaf tissue were cut from the edge of the lesions, surface sterilized and placed on potato dextrose agar (PDA) medium amended with kanamycin (0.1 mg/ml). Isolate J136, one of five isolates with similar morphology, was selected for pathogen identification. The culture of the isolate on PDA was brown and exhibited radial mycelial growth after incubation at 28 oC in darkness for 5 days. Hyphae of the fungus were white at the beginning, turned light brown to brown at the later stages, and finally became thick and separated. Sclerotia were brown and produced on PDA after 25 days of incubation in the dark. These characteristics were similar to the colony characteristics of R. solani. The genomic DNA of Isolate J136 was extracted using the CTAB method. PCR analyses were conducted using the following primers specifically designed for the detection of individual AGs or subgroups of R. solani: AG-1 IA, IB and IC (Kuninaga 2003), AG-2-1, AG-2-2, IIIB, IV and LP (Carling et al. 2002), AG-3 PT (Misawa 2015), AG-4 HG-I and HG-II (Kuninaga 2003), and AGs-5-6 (Arakawa and Inagaki 2014). Among the 12 specific primer pairs, only AG-6-specific primers amplified a fragment of ca. 230 bp product, indicating that the tested strain belonged to R. solani AG-6. The sequence was deposited in GenBank with accession no. MZ379468. Using BLASTN search, the sequence of the gene was aligned with the voucher specimen, R. solani AG-6. A phylogenetic tree was constructed based on these sequences. After wards, Isolate J136 was tested for hyphal anastomosis reaction using the R. solani AG-6 standard strain according to the method described by Ogoshi (1987). The hyphal diameter at the point of anastomosis was reduced, with obvious anastomosis point, and the death of adjacent cells, indicating their anastomosis reactions (Anderson 1982). Thus, based on the morphological and genetic analyses, the fungus was identified as R. solani AG-6. To verify its pathogenicity, six plants (cv. Yunyan87) at the 5-to-6 leaf stage were inoculated with mycelial PDA plugs (5 mm in diameter). Leaves inoculated with PDA-only plugs served as the controls. Treated tobacco plants were maintained at a temperature range of 15 to 25 oC in a greenhouse with 85% relative humidity. After 5 days inoculation, typical symptoms were observed on the inoculated leaves, whereas no symptoms were observed on the control leaves. Koch's postulates were fulfilled by re-isolation of the pathogen from the diseased leaves. R. solani AG-2-2 is the only previously reported group of R. solani, which causes tobacco target spot in the field (Gonzalez et al. 2011). Therefore, to our knowledge, this is the first report of R. solani AG-6 causing target spot of tobacco in the field in China. Since considerable losses caused by the disease have frequently happened in this region, addition of this new group pathogen in the disease pool can be more problematic. Proper disease control strategies are in need to be developed to prevent further losses.

New Anastomosis Group F (AG-F) of binucleate Rhizoctonia causing root and stem rot of Pistacia vera

A total of ten isolates of fungi with Rhizoctonia-like mycelia were obtained from infected roots and stems of Pistachio Pistacia vera grown in a commercial nursery in Rafsanjan, Iran, during the autumn of 2011. The infected seedlings showed symptoms of chlorosis that later turned to necrosis. All of the isolates were identified as binucleate Rhizoctonia on the basis of hyphal characteristics and nuclear number. They were tested for detection of the anastomosis group, optimum growth temperature, rDNA-ITS region traits and pathogenicity on pistachio seedlings in vitro and in vivo. The analysis of hyphal anastomosis reaction was carried out with the tester isolates of binucleate Rhizoctonia AG-A, AG-Ba, AG-G and AG-F as well as multinucleate Rhizoctonia AG4 as already detected on pistachio seedlings. The optimum temperature for growth of binucleate Rhizoctonia sp.was 35 °C. In in vivo test, the symptoms of root rot were observed 30 days after inoculation and mortality happened two weeks thereafter. According to molecular characteristics and anastomosis test groups, all isolates showed greatest similarity to anastomosis group AG-F. This finding is the first report of anastomosis group F (AG-F) of binucleate Rhizoctonia, as causal agent of root and stem rot disease of pistachio in the world and Iran.

Characterization of Rhizoctonia solani Associated with Black Scurf in Cyprus.

During 2011, 96 sclerotial isolates of Rhizoctonia solani were collected from potato tubers from all main potato-cultivating regions of Cyprus. All isolates were found to be multinucleate. Characterization of anastomosis groups (AG) based on hyphal anastomosis reactions showed that 91 isolates belonged to AG3 and 5 to AG4. Sequence analysis of the internal transcribed spacer (ITS) regions (ITS1 and ITS2) of ribosomal DNA (rDNA) of 68 isolates confirmed the prevalence of AG3. In addition, phylogenetic analysis found that AG3 isolates were of the potato type, distinctly separated from the AG3 tobacco type, while AG4 isolates were separated into two different subgroups (HGI and HGII). Temperature studies showed that isolates belonging to both AG4 subgroups had significantly higher optimum growth temperatures compared with AG3. In vitro sensitivities to the fungicide pencycuron, in terms of concentrations where 50% growth inhibition was observed, ranged from 0.012 to 0.222 μg/ml. Pathogenicity and aggressiveness of the isolates was determined on 'Annabelle' potato sprouts and seedlings of a number of selected hosts, based on crop rotations followed in Cyprus. The majority of the isolates were pathogenic to potato sprouts, with disease severity (DS) values ranging from 0 to 88%. Mean DS values were statistically different among AG and subgroups, with AG4-HGI (69.25%) and AG4-HGII (3.12%) being the most and least aggressive, respectively. However, AG4-HGII isolates were the most aggressive in all rotational hosts tested, while AG3 isolates were the least aggressive. More specifically, the highest DS levels by AG4-HGI were recorded to barley, by AG4-HGII to lettuce and melon, and by AG3 isolates to vetch. This is the first comprehensive study to elucidate the AG composition, pathogenicity and other biological aspects of R. solani isolates associated with potato black scurf in Cyprus.

First Report of Web Blight on Rose Campion (Lychnis coronaria) Caused by Rhizoctonia solani AG 1-IB in Italy.

Lychnis coronaria (syn. Silene coronaria), rose campion, is a perennial in the Caryophyllaceae used in gardens. In the summer of 2014, a web blight was observed in a private garden located near Biella (northern Italy), approximately 45°39'N 8°00'E, on 40% of 100 5-month-old plants grown in sandy soil. In the days preceding the outbreak of the disease, daytime temperatures ranged from 18 to 24°C and relative humidity from 45 to 83%. Affected plants showed pale brown discoloration of stems, starting from the base, and eventually collapsed. Under conditions of high relative humidity, a scant amount of whitish mycelium developed on leaves of about 50% of diseased plants. Eventually, infected plants died about 10 days after symptoms appeared. Symptomatic tissues of stems and leaves were disinfected for 10 s in 1% NaOCl, rinsed in sterile water, and plated on potato dextrose agar (PDA). A fungus with the morphological characters of Rhizoctonia solani (3) was consistently recovered. Three representative isolates were paired with tester strains of R. solani (AG 1, AG 2-2-IIIB, AG 4, AG 7, and AG 11) (2) and examined microscopically. The Lychnis isolates anastomosed only with the AG 1 tester strain, with low fusion frequency. The anastomosis point was obvious: the hyphal diameter at the point of anastomosis was reduced and death of adjacent cells was observed, indicating an anastomosis reaction (1). Mycelium maintained on PDA at 23 ± 1°C was coarse and reddish brown. After 5 days of growth, mycelium started differentiating numerous sclerotia, often aggregated. Mature sclerotia were dark, spheroidal, with diameters ranging from 0.2 to 1.6 (average 0.6) mm. The internal transcribed spacer (ITS) region of rDNA was amplified using the primers ITS1/ITS4 and sequenced. BLASTn analysis of the 609-bp amplicon (GenBank Accession No. KM596899) showed a 98% homology with the sequence of the R. solani isolate FJ746937 obtained from Zoysiagrass. On the basis of molecular and cultural characteristics and anastomosis tests, the isolates from L. coronaria were identified as R. solani AG 1-IB (4). For pathogenicity tests performed in August, mycelial disks (8 mm diam.) from 10-day-old PDA cultures of an isolate of the fungus were placed on four healthy 6-month-old L. coronaria plants (four stem and six leaf disks per plant). Four plants inoculated with disks of PDA served as controls. Plants were covered with plastic bags for 4 days and maintained in a garden located in the same area in which the disease appeared, at field temperatures ranging from 15 to 28°C. The first symptoms developed 4 days after inoculation, and 15 days after the artificial inoculation, all inoculated plants were dead. R. solani was re-isolated from the stem of symptomatic plants, whereas no colonies developed from controls, which all remained healthy. This is the first report of blight of L. coronaria caused by R. solani in Italy or anywhere else in the world. The impact of this disease may become a significant problem for L. coronaria, a very common species in Italian gardens.

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

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

Molecular markers for genotyping anastomosis groups and understanding the population biology of Rhizoctonia species

Soil-borne Rhizoctonia fungi cause serious diseases in several plant species. For the classification of these fungi, the number of nuclei in a hyphal cell and the anastomosis reaction are important criteria. Although Rhizoctonia spp. has a wide host range, the causal agents have been reported to be selective for host plant families or species and lead to severe disease. Reports of new diseases, particularly in new host plants, and severe damage in agricultural fields incurred by subdivided or newly found groups of Ceratobasidium and Waitea circinata (a varied teleomorph of Rhizoctonia) have been increasing in recent years. The food production environment is altering because of climate change, introduction of potential new host plants, and heavy use of chemicals that reduce microbial diversity. These changes favor the occurrence of new diseases incurred by undefined anastomosis groups (AGs) or subgroups of Rhizoctonia spp. On the basis of the phylogenetic relationships of AGs and subgroups in Rhizoctonia spp., molecular markers for discriminating the groups of the Rhizoctonia species complex have been developed. The application of genetic markers, in the form of microsatellites or simple sequence repeats (SSR), has become increasingly important in fungal genetics. The analyses of population genetics for Rhizoctonia spp. using SSR markers elucidated the modes of sexual and asexual reproduction, phylogeographical distributions, and global migrations associated with adaptation to agroecosystems.

First Report of Web Blight on Rebutia perplexa Caused by Rhizoctonia solani AG 2-2-IIIB in Italy.

Rebutia perplexa, Cactaceae family, is a clumping fine thorny cactus, producing several flushes of pink flowers. In the spring of 2013, a blight was observed in a farm located near Imperia (northern Italy) on 2% of 2,000 3-year-old plants, grown in plastic pots. Affected plants showed pale brown discoloration of stems, starting from the base, and eventually collapsed. Flowers also rotted and wilted. In the presence of high relative humidity, a rare, whitish mycelium developed on the surface of the substrate. Eventually, infected plants died. Symptomatic tissues of the stem were taken from 10 plants and plated on potato dextrose agar (PDA). A fungus with the morphological characters of Rhizoctonia solani (3) was consistently recovered. Three representative isolates obtained from affected plants were successfully paired with tester strains of R. solani (AG 1, AG 2-2-IIIB, AG 2-2-IV, AG 4, AG 7, AG 11) (2) and examined microscopically. Three replicated pairings were made for each tester strain. The Rebutia isolates anastomosed only with AG 2-2-IIIB tester strain with high hyphal fusion frequency. The hyphal diameter at the point of anastomosis was reduced, the anastomosis point was obvious, and death of adjacent cells was observed, indicating anastomosis reactions (1). Tests were performed twice. Mycelium of 15-day-old isolates maintained at 27 to 30°C, appeared whitish or pale buff in color, coarse, with a concentric zonation, scarce aerial mycelium, and without sclerotia. The optimum temperature for mycelium growth was 30°C (daily growth rate: 24.6 mm) and isolates grew also at 35°C. The internal transcribed spacer (ITS) region of rDNA was amplified using the primers ITS1/ITS4 and sequenced. BLASTn analysis of the 523-bp amplicon (GenBank Accession No. KF719318) showed a 99% homology with the sequence of the R. solani AG 2-2-IIIB isolate GU811672. The nucleotide sequence has been assigned the GenBank Accession No. KF719318. Therefore, on the basis of molecular characteristics, anastomosis tests, temperature growth, and cultural characteristics, the isolates from R. perplexa were identified as R. solani AG 2-2-IIIB. For pathogenicity tests, 3 g of colonized wheat kernel from 10-day-old cultures of a representative isolate of the fungus was added per 1 l of substrate in 12 potted healthy plants of R. perplexa. The inoculum was prepared by inoculating wheat kernels with the mycelium of 10-day-old cultures of the fungus and incubating at 25 ± 1°C (12 h fluorescent light, 12 h dark). Twelve plants inoculated with non-infested wheat kernels served as controls. Plants were covered with plastic bags and maintained in a growth chamber at 25 ± 1°C. The first symptoms, similar to those observed in the farm, developed 5 days after inoculation. Fifteen days after the artificial inoculation, all inoculated plants were dead. R. solani was re-isolated only from the stems of symptomatic plants. Control plants remained healthy. The pathogenicity test was carried out twice with similar results. This is, to our knowledge, the first report of blight of R. perplexa caused by R. solani in Italy as well as worldwide.

Rhizoctonia属菌における菌糸融合群判定および集団遺伝学解析のための分子マーカー

Soil-borne Rhizoctonia fungi cause serious diseases in several plant species. For the classification of these fungi, the number of nuclei in a hyphal cell and the anastomosis reaction are important criteria. Although Rhizoctonia spp. has a wide host range, the causal agents have been reported to be selective for host plant families or species and lead to severe disease. Reports of new diseases, particularly in new host plants, and severe damage in agricultural fields incurred by subdivided or newly found groups of Ceratobasidium and Waitea circinata (a varied teleomorph of Rhizoctonia) have been increasing in recent years. The food production environment is altering because of climate change, introduction of potential new host plants, and heavy use of chemicals that reduce microbial diversity. These changes favor the occurrence of new diseases incurred by undefined anastomosis groups (AGs) or subgroups of Rhizoctonia spp. On the basis of the phylogenetic relationships of AGs and subgroups in Rhizoctonia spp., molecular markers for discriminating the groups of the Rhizoctonia species complex have been developed. The application of genetic markers, in the form of microsatellites or simple sequence repeats (SSR), has become increasingly important in fungal genetics. The analyses of population genetics for Rhizoctonia spp. using SSR markers elucidated the modes of sexual and asexual reproduction, phylogeographical distributions, and global migrations associated with adaptation to agroecosystems.

First Report of Aerial Blight of Peanut Caused by Rhizoctonia solani AG1-IA in Arkansas.

In early September 2012, symptoms similar to aerial blight were observed on runner peanut (cv. Georgia 09B) in a commercial field in Randolph County, Arkansas (3). Leaves within the canopy closest to the soil had water-soaked, gray to green lesions or tan to brown lesions. Localized areas of matted leaves with mycelium occurred on stems and hyphae extended along stems and newly affected leaves. Dark brown spherical sclerotia (1.5 to 4 mm diam.) were produced on the surface of symptomatic peanut tissue (3). Aerial blight symptoms were observed in two peanut fields (~4 to 6 ha) that were furrow irrigated. Symptomatic plants were localized in a single circular pattern (~20 × 25 m) near the lower end of each field with the final disease incidence of less than 5%. Isolations from surface-disinfected leaves on potato dextrose agar consistently yielded light brown to brown colonies with sclerotia typical of Rhizoctonia solani AG1-IA. The fungus was confirmed to be R. solani AG1 by anastomosis reaction (2) with known cultures of AG1-IA isolated from soybean and rice in Arkansas. Sequencing of the rDNA ITS region 5.8s with primers ITS1 and ITS4 (1) supported the identification of the R. solani isolates as AG1-IA. The BLAST search revealed that the sequence had a 96 to 97% maximum sequence identity to several R. solani AG1-IA isolates collected from rice sheaths in China and Arkansas. Eight-week-old peanut plants (cv. Georgia 09B) growing in pots were sprayed until runoff (2 ml/plant) with a solution containing approximately 1 × 105 hyphal fragments/ml. Five inoculated plants were placed in a humidity chamber within a greenhouse where temperatures ranged from 28 to 33°C. After 14 days, water soaked, gray to green or light brown lesions developed on all inoculated plants along with hyphal strands along inoculated sections of the peanut with dark brown sclerotia. None of the plants inoculated with sterile water expressed symptoms. Rhizoctonia solani was consistently reisolated from symptomatic tissue plated on PDA. Inoculations were repeated on peanut cv. Flavor Runner 458, Florida 07, FloRun 107, and Red River Runner with similar results. Although R. solani AG1-IA is a common pathogen on rice and soybean, causing sheath blight and aerial blight, respectively, to our knowledge this is the first report of aerial blight of peanut in the region. Currently, there is a renewed interest in peanut production in the state. Production practices include furrow irrigation, which can distribute floating sclerotia to peanut vines and the rotation practiced with soybean and, less frequently, rice, could potentially increase inoculum for the subsequent crop. Thus, this may be a significant disease problem in the region or Mid-South where peanut is planted after rice or soybean and furrow irrigated.

Progeny derived from Thanatephorus cucumeris (Rhizoctonia solani) AG-2-2 IV isolates on sugar beet foliage show more clonal diversity in somatic compatibility grouping than those from roots and petioles

Twenty-nine isolates of Thanatephorus cucumeris (Rhizoctonia solani) AG-2-2 IV were collected from the roots, petioles, and leaves of diseased sugar beets in Hokkaido, Japan. We examined the genetic variation of the field isolates using somatic compatibility grouping of progeny (cultured isolates derived from induced basidiospores) based on analysis of hyphal anastomosis reactions (i.e., hyphal perfect fusion) and inter-simple sequence repeats (ISSRs). The number of somatic compatibility groups (SCGs) of single basidiospore isolates was strongly correlated with the host plant tissue from which the parental isolates were obtained. Parental isolates from roots and petioles tended to be genetically heterogeneous and generated plural SCGs, whereas isolates (except for two non-self-anastomosing isolates) from leaves tended to be homogeneous and generated a single SCG. Heterogeneous sibling basidiospore isolates yielded homogeneous progeny within a few generations. These findings were supported by the results of ISSR analysis, which showed that the homogeneous isolates generated progeny with the same genotype, whereas heterogeneous isolates generated progeny with different genotypes. However, like SCGs, ISSR genotypes of heterogeneous progeny tended to be homogeneous within two or three generations. Additionally, we examined clonal diversity during the basidiospore infection process over a 2-year period. A heterogeneous isolate generated a large number of progeny SCGs, thereby increasing clonal diversity. In contrast, progeny SCGs were almost all the same in a homogeneous isolate plot with a few exceptions. These results indicate that infection of T. cucumeris basidiospores play a role in the clonal diversity of AG-2-2 IV isolates, as indicated by progeny SCGs in the field.

English

Rhizoctonia solani is considered as one of the most destructive soil-and-seed borne plant pathogens infecting various agricultural crops including chickpea. The 50 chickpea isolates of R. solani representing 10 different states of India were variable in hyphal anastomosis reactions and they were grouped into seven AGs as AG1, AG2-2, AG2-2LP, AG2-3, AG3, AG4 and AG5. Genetic diversity of the pathogen was determined by using molecular markers namely, simple sequence repeats (SSR) and rDNA internal transcribed spacer (ITS). A neighbor-joining tree constructed based on the profiles generated by SSR markers grouped the isolates into eight categories. This revealed 90% of genetic similarity among the isolates and partial correlation with reference to their geographical origin and AGs. The isolates were amplified with a set of primers ITS 1 and ITS 4 and they produced a specific band ≈650 bp. Low level of (7%) variability was observed in the nucleotide sequences of the ITS regions of these isolates. The phylogenic tree generated from bootstrap neighboring joint analysis grouped the Indian populations of R. solani into two categories.   Key words: Chickpea, wet root rot, genetic diversity, simple sequence repeats (SSR), internal transcribed spacer (ITS).

DNA fingerprinting and anastomosis grouping reveal similar genetic diversity in Rhizoctonia species infecting turfgrasses in the transition zone of USA

Rhizoctonia blight is a common and serious disease of many turfgrass species. The most widespread causal agent, Thanatephorus cucumeris (anamorph: R. solani), consists of several genetically different subpopulations. In addition, Waitea circinata varieties zeae, oryzae and circinata (anamorph: Rhizoctonia spp.) also can cause the disease. Accurate identification of the causal pathogen is important for effective management of the disease. It is challenging to distinguish the specific causal pathogen based on disease symptoms or macroscopic and microscopic morphology. Traditional methods such as anastomosis reactions with tester isolates are time consuming and sometimes difficult to interpret. In the present study universally primed PCR (UP-PCR) fingerprinting was used to assess genetic diversity of Rhizoctonia spp. infecting turfgrasses. Eighty-four Rhizoctonia isolates were sampled from diseased turfgrass leaves from seven distinct geographic areas in Virginia and Maryland. Rhizoctonia isolates were characterized by ribosomal DNA internal transcribed spacer (rDNA-ITS) region and UP-PCR. The isolates formed seven clusters based on ITS sequences analysis and unweighted pair group method with arithmetic mean (UPGMA) clustering of UP-PCR markers, which corresponded well with anastomosis groups (AGs) of the isolates. Isolates of R. solani AG 1-IB (n = 18), AG 2-2IIIB (n = 30) and AG 5 (n = 1) clustered separately. Waitea circinata var. zeae (n = 9) and var. circinata (n = 4) grouped separately. A cluster of six isolates of Waitea (UWC) did not fall into any known Waitea variety. The binucleate Rhizoctonia-like fungi (BNR) (n = 16) clustered into two groups. Rhizoctonia solani AG 2-2IIIB was the most dominant pathogen in this study, followed by AG 1-IB. There was no relationship between the geographic origin of the isolates and clustering of isolates based on the genetic associations. To our knowledge this is the first time UP-PCR was used to characterize Rhizoctonia, Waitea and Ceratobasidium isolates to their infra-species level.

First Report of Web Blight on Oregano (Origanum vulgare L.) Caused by Rhizoctonia solani AG-1-IB in Italy.

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.

First Report of Web Blight on Rosemary (Rosmarinus officinalis) Caused by Rhizoctonia solani AG-1-IA in Italy.

Rosmarinus officinalis L., family Labiatae, is an evergreen shrub used in gardens as an aromatic or ground cover plant. In the summer of 2012, a blight was observed in a farm located near Albenga (northern Italy) on 20% of 150,000 70-day-old plants, grown in trays. Water soaked lesions appeared on leaves and stems. As the disease progressed, blighted leaves turned brown, withered, clung to the shoots, and matted on the surrounding foliage. A light mycelium spread on the substrate. Disease progressed from infected plants to healthy ones and, 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 and readily recovered. Three isolates of R. solani obtained from affected plants were successfully paired with R. solani tester strains AG 1, 2, 3, 4, 6, 7, or 11 and examined microscopically. Three pairings were made for each recovered isolate. The isolates of R. solani from rosemary anastomosed only with tester strain AG 1 (ATCC 58946). Results were consistent with other reports on anastomosis reactions (2). Tests were repeated once. Mycelium of 10-day-old isolates from rosemary appeared light brown, compact, and radiate. Numerous dark brown sclerotia, 0.7 to 2.0 mm diameter (average 1.3), developed within 10 days at 20 to 26°C. The descriptions of mycelium and sclerotia were typical for subgroup IA Type 2 (4). The internal transcribed spacer (ITS) region of rDNA was amplified using the primers ITS1/ITS4 and sequenced (GenBank Accession No. KC005724). BLASTn analysis (1) of the 657-bp showed a 99% similarity with the sequence of R. solani GU596491. For pathogenicity tests, inoculum of R. solani was prepared by growing the pathogen on wheat kernels autoclaved in 1-liter glass flasks for 8 days. One of the isolates assigned to the anastomosis group AG 1 IA was tested. Fifteen 90-day-old rosemary plants were grown in 15-liter pots in a steam disinfested peat:pomice:pine bark:clay mix (50:20:20:10) infested with 3 g/liter of infested wheat kernels, placed at the base of the stem. Fifteen plants inoculated with non-infested wheat kernels served as control treatments. Plants were covered with plastic bags and arranged in a growth chamber at 20 to 24°C with 12 h light/dark for 15 days. The first symptoms, similar to those observed in the farm, developed 10 days after inoculation. About 10 colonies of R. solani were reisolated from infected leaves and stems of each inoculated plant. Control plants remained healthy. The pathogenicity test was carried out twice with similar results. Symptoms caused by R. solani have been recently observed on R. officinalis in United States (3), India, and Brazil. This is, to our knowledge, the first report of blight of R. officinalis caused by R. solani in Italy. This disease could cause serious economic losses, because rosemary is one of the most cultivated aromatic plants in the Mediterranean region.

First Report of Web Blight on Winter Savory (Satureja Montana "Repandens") Caused by Rhizoctonia solani AG-1-IA in Italy.

Satureja montana L. (winter savory "Repandens") is an evergreen shrub. In late summer 2010, blight was observed on a farm near Albenga (northern Italy) on 3% of 500 potted 2-month-old plants. Semicircular, water-soaked lesions appeared first on stems then on leaves. As the disease progressed, blighted leaves turned brown, withered, clung to the shoots, and matted on the surrounding foliage within 5 to 6 days. Stem fragments taken from the margin of the diseased tissues of 10 plants were disinfected for 10 s in 1% NaOCl, rinsed with sterile water, and plated on potato dextrose agar (PDA) amended with 100 μg/liter streptomycin sulfate. A fungus with morphological characters of Rhizoctonia solani was consistently isolated. 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 tested strain. 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 winter savory 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 repeated once. Mycelium of 10-day-old isolates from winter savory appeared light brown, compact, and radiate. Numerous, dark brown sclerotia, 1 to 4 mm in diameter (average 1.7), developed within 20 days after transfer of mycelia to PDA in 90-mm-diameter petri dishes and incubated (11-h daylight, 13-h dark) at 21 to 24°C. Descriptions of mycelium and sclerotia were typical for subgroup IA Type 2 (3). The internal transcribed spacer (ITS) region of rDNA was amplified with primers ITS1/ITS4 and sequenced. BLASTn analysis (1) of the 696 bp showed a 99% homology with the sequence of R. solani. The nucleotide sequence has been assigned GenBank No. JQ313811. For pathogenicity tests, inoculum of R. solani was prepared by growing the pathogen on wheat kernels autoclaved in 1-liter glass flasks (30 min at 121°C and 1 atm) for 15 days. One of the isolates assigned to the anastomosis group AG 1 IA was tested. Five 90-day-old plants of S. montana were inoculated. Each plant grown in 2-liter pots in a steam disinfested peat/pumice/pine bark/clay mix (50:20:20:20:10) was inoculated with 10 g of infested wheat kernels placed at the base of the stem. Five plants inoculated with noninfested wheat kernels served as the control. Plants covered with plastic bags were arranged randomly in a growth chamber at 20 ± 1°C with 12-h light/dark for 5 days. Symptoms, similar to those observed in the farm, developed 4 days after inoculation. Ten colonies of R. solani were reisolated from infected leaves and stems of each inoculated plant. Control plants remained healthy. The pathogenicity test was carried out twice. Symptoms caused by R. solani have been recently observed on S. hortensis in Poland (4). This is, to our knowledge, the first report of blight of S. montana caused by R. solani in Italy.

Characterization of Rhizoctonia spp. Isolates Associated with Damping-Off Disease in Cotton and Tobacco Seedlings in Greece.

Isolates of Rhizoctonia spp. were obtained during the spring of 2007 from diseased cotton and tobacco seedlings showing damping-off symptoms. The sampled fields were located in the main cotton- and tobacco-cultivating regions of central and northern Greece. Among the 79 isolates obtained from cotton plants, 17 were binucleate and 62 were multinucleate whereas, among the 89 isolates obtained from tobacco plants, 87 were multinucleate and only 2 were binucleate. Characterization of anastomosis groups (AGs) was performed with hyphal anastomosis reactions using tester isolates of known AG groups. Anastomosis reactions in cotton mutlinucleate isolates showed that 54 of them belonged in Rhizoctonia solani AG-4, 6 in AG-7, 1 in AG-2, and 1 in AG-3. In the 87 tobacco multinucleate isolates, anastomosis reactions showed that 70 of them belonged in R. solani AG-2, 16 in AG-4, and 1 in AG-5. In addition, molecular characterization was carried out using the specific ribosomal DNA internal transcribed spacer region, in a randomly selected number of isolates. In cotton, the most prevalent AG was AG-4, with 18 isolates to the subgroup HG-I, 1 isolate to the subgroup HG-II, and 7 isolates to the subgroup HG-III, followed by AG-7 (7 isolates), AG-2-1 (1 isolate), and AG-3 (1 isolate). In tobacco, the most prevalent group was AG-2-1 (70 isolates), followed by AG-4 (6 isolates to the subgroup HG-I and 5 isolates to the subgroup HG-III) and a single isolate belonging to AG-5. Phylogenetic analysis showed that the isolates were distinctly separated based on their AG types. Pathogenicity and aggressiveness of the isolates to several hosts was determined. AG-4 isolates from either cotton or tobacco were the most aggressive on the hosts tested, while AG-2-1 isolates were of moderate aggressiveness and were not pathogenic on barley.

Tobacco leaf spot and root rot caused by Rhizoctonia solani Kühn

Rhizoctonia solani Kühn is a soil-borne fungal pathogen that causes disease in a wide range of plants worldwide. Strains of the fungus are traditionally grouped into genetically isolated anastomosis groups (AGs) based on hyphal anastomosis reactions. This article summarizes aspects related to the infection process, colonization of the host and molecular mechanisms employed by tobacco plants in resistance against R. solani diseases. Teleomorph: Thanatephorus cucumeris (Frank) Donk; anamorph: Rhizoctonia solani Kühn; Kingdom Fungi; Phylum Basidiomycota; Class Agaricomycetes; Order Cantharellales; Family Ceratobasidiaceae; genus Thanatephorus. Somatic hyphae in culture and hyphae colonizing a substrate or host are first hyaline, then buff to dark brown in colour when aging. Hyphae tend to form at right angles at branching points that are usually constricted. Cells lack clamp connections, but possess a complex dolipore septum with continuous parenthesomes and are multinucleate. Hyphae are variable in size, ranging from 3 to 17 µm in diameter. Although the fungus does not produce any conidial structure, ellipsoid to globose, barrel-shaped cells, named monilioid cells, 10-20 µm wide, can be produced in chains and can give rise to sclerotia. Sclerotia are irregularly shaped, up to 8-10 mm in diameter and light to dark brown in colour. Symptoms in tobacco depend on AG as well as on the tissue being colonized. Rhizoctonia solani AG-2-2 and AG-3 infect tobacco seedlings and cause damping off and stem rot. Rhizoctonia solani AG-3 causes 'sore shin' and 'target spot' in mature tobacco plants. In general, water-soaked lesions start on leaves and extend up the stem. Stem lesions vary in colour from brown to black. During late stages, diseased leaves are easily separated from the plant because of severe wilting. In seed beds, disease areas are typically in the form of circular to irregular patches of poorly growing, yellowish and/or stunted seedlings. Knowledge is scarce regarding the mechanisms associated with resistance to R. solani in tobacco. However, recent evidence suggests a complex response that involves several constitutive factors, as well as induced barriers controlled by multiple defence pathways. This fungus can survive for many years in soil as mycelium, and also by producing sclerotia, which makes the management of the disease using conventional means very difficult. Integrated pest management has been most successful; it includes timely fungicide applications, crop rotation and attention to soil moisture levels. Recent developments in biocontrol may provide other tools to control R. solani in tobacco.

First Report of Leaf Blight on Woodland Sage Caused by Rhizoctonia solani AG 1 in Italy.

Woodland sage (Salvia nemorosa L.; Lamiaceae) is a hardy herbaceous perennial plant that is easy to grow and propagate and is used in parks and grown as potted plants. During the summer of 2009 in a nursery near Torino in northern Italy, a leaf blight was observed on 30-day-old plants of cv. Blau Koenigin grown in pots under shade. Semicircular, water-soaked lesions developed on leaves just above the soil line at the leaf-petiole junction and later along leaf margins. Lesions expanded along the midvein until the entire leaf was destroyed. Blighted leaves turned brown, withered, and clung to the shoots. No symptoms were observed on the roots. Severely infected plants died. Diseased tissue was disinfested for 10 s in 1% NaOCl, rinsed with sterile water, and plated on potato dextrose agar (PDA) amended with 25 mg/liter of streptomycin sulfate. A fungus with morphological characters of Rhizoctonia solani (3) was consistently recovered. Ten-day-old mycelium grown on PDA at 22 ± 1°C appeared light brown, rather compact, and with radial growth. Sclerotia were irregular and measured between 0.5 and 2 mm. Pairings were made with tester isolates of AG 1, 2, 3, 4, 5, 6, 7, 11, and AG B1. The only successful anastomosis was with tester isolate AG 1 (ATCC 58946). The hyphal diameter at the point of anastomosis was reduced, the anastomosis point was obvious, and cell death of adjacent cells was observed. Results were consistent with other reports on anastomosis reactions (2). The description of sclerotia of the isolate AG1 was typical for subgroup 1A Type 2 (3). The internal transcribed spacer (ITS) region of rDNA was amplified using primers ITS4/ITS6 and sequenced. BLASTn analysis (1) of the 688 bp showed a 100% homology with the sequence of R. solani AG-1A and the nucleotide sequence has been assigned (GenBank Accession No. HM044764). For pathogenicity tests, the inoculum of one isolate of R. solani from the nursery was prepared by growing the pathogen on PDA for 7 days. The foliage of 30-day-old potted plants of S. nemorosa cv. Blau Koenigin was artificially inoculated with an aqueous suspension of PDA and mycelium fragments (1 g per mycelium per plant) prepared from cultures with a blender. Plants were covered with plastic bags for 3 days. Plants inoculated with water and PDA fragments alone served as control treatments. Plants were maintained in a glasshouse at 20 to 25°C. The first symptoms, similar to those observed in the nursery, developed 7 days after foliar inoculation. R. solani was consistently reisolated from infected leaves. Control plants remained healthy. The pathogenicity test was carried out twice with similar results. To our knowledge, this is the first report of leaf blight of S. nemorosa caused by R. solani in Italy as well as worldwide. The importance of the disease is still unknown.