Solani Anastomosis Group Research Articles

Development of a Greenhouse Assay to Screen Soybean Varieties for Resistance to Aerial Blight Caused by Rhizoctonia solani Anastomosis Group 1-IA.

Aerial blight, caused by the fungus Rhizoctonia solani anastomosis group (AG) 1-IA, is an economically important soybean disease in the mid-Southern United States. Management has relied on fungicide applications during the season, but there is an increasing prevalence of resistance to commonly used strobilurin fungicides and an urgent need to identify soybean varieties resistant to aerial blight. Because the patchy distribution of the pathogen complicates field variety screening, the present study aimed to develop a greenhouse screening protocol to identify soybean varieties resistant to aerial blight. For this, 88 pathogen isolates were collected from commercial fields and research farms across five Louisiana parishes, and 77% were confirmed to be R. solani AG1-IA. Three polymorphic codominant microsatellite markers were used to explore the genetic diversity of 43 R. solani AG1-IA isolates, which showed high genetic diversity, with 35 haplotypes in total and only two haplotypes common to two other locations. Six genetically diverse isolates were chosen and characterized for their virulence and fungicide sensitivity. The isolate AC2 was identified as the most virulent and was resistant to both active ingredients, azoxystrobin and pyraclostrobin, tested. The six isolates were used in greenhouse variety screening trials using a millet inoculation protocol. Of the 31 varieties screened, only Armor 48-D25 was classified as moderately resistant, and plant height to the first node influenced final disease severity. The study provides short-term solutions for growers to choose less susceptible varieties for planting and lays the foundation to characterize host resistance against this important soybean pathogen.

Disentangling Shifts in the Soil Microbiome of Potatoes Infected with Rhizoctonia solani Anastomosis Group 3-PT in Search of Potential Biocontrol Agents

Rhizoctonia solani anastomosis group (AG) 3-PT is a devastating pathogenic fungus that causes several diseases in potatoes both in South Africa and globally. The removal of various fungicides from the market and strict regulations on the use of synthetic chemicals make disease management difficult. Therefore, alternative, environmentally safe control measures are being considered, such as the use of biological control agents (BCAs). BCAs are an attractive alternative for improving plant and soil health of economically important crops. To identify key microbial indicators of disease suppression against R. solani AG 3-PT, a greenhouse pot trial experiment was conducted using soil from a potato-growing region in KwaZulu-Natal, South Africa. High-throughput sequencing of fungal internal transcribed spacer and bacterial 16S ribosomal RNA was used to characterize the respective fungal and bacterial community composition in the soil with and without artificial inoculation with R. solani AG 3-PT. Results indicated that the pathogen caused dysbiosis in the potato soil microbiome, leading to a shift in fungal and bacterial community composition. Differentially abundant microbial taxa in R. solani AG 3-PT inoculated soils suggest a promising potential for disease-suppressive activity. Network analysis also confirmed the presence of key taxa involved in the microbial community shifts, which could support their role in the suppression of R. solani AG 3-PT. The identification of key microbial indicators against Rhizoctonia diseases can contribute to the development of environmentally sustainable potato production systems, which are particularly important considering the implementation of the European Green Deal.

Unveiling the Genetic Tapestry: Exploring Rhizoctonia solani AG-3 Anastomosis Groups in Potato Crops across Borders.

The current study was carried out to screen 10 isolates (ARS-01-ARS-10) of Rhizoctonia. solani from potato tubers cv. Kuroda, which were collected from various potato fields in Multan, Pakistan. The isolates were found to be morphologically identical, as the hyphae exhibit the production of branches at right angles and acute angles often accompanied by septum near the emerging branches. Anastomosis grouping showed that these isolates belonged to AG-3. A pathogenicity test was performed against the susceptible Kuroda variety and among the isolates, ARS-05 exhibited the highest mean severity score of approximately 5.43, followed by ARS-09, which showed a mean severity score of about 3.67, indicating a moderate level of severity. On the lower end of the severity scale, isolates ARS-06 and ARS-07 displayed mean severity scores of approximately 0.53 and 0.57, respectively, suggesting minimal symptom severity. These mean severity scores offer insights into the varying degrees of symptom expression among the different isolates of R. solani under examination. PCoA indicates that the severe isolate causing black scurf on the Kuroda variety was AG-3. A comprehensive analysis of the distribution, genetic variability, and phylogenetic relationships of R. solani anastomosis groups (AGs) related to potato crops across diverse geographic regions was also performed to examine AG prevalence in various countries. AG-3 was identified as the most widespread group, prevalent in Sweden, China, and the USA. AG-5 showed prominence in Sweden and the USA, while AG-2-1 exhibited prevalence in China and Japan. The phylogenetic analysis unveiled two different clades: Clade I comprising AG-3 and Clade II encompassing AG-2, AG-4, and AG-5, further subdivided into three subclades. Although AGs clustered together regardless of origin, their genetic diversity revealed complex evolutionary patterns. The findings pave the way for region-specific disease management strategies to combat R. solani's impact on potato crops.

First Report of Root and Bulb Rot Caused by Rhizoctonia solani AG-6 on Lanzhou Lily (Lilium davidii var. unicolor) in China.

Lanzhou lily (Lilium davidii var. unicolor) is the only famous sweet lily variety that has high edible, medicinal and ornamental value in China, which is mostly planted in the middle areas of Gansu Province in China. In recent years, severe yellowing and wilting of leaves, stem wilt, root and bulb rot symptoms were observed on Lanzhou lily in Qilihe District, Lanzhou, which has resulted in serious loss of bulb production. From June to August 2022, a survey of Lanzhou lily disease was carried out in Xiguoyuan and Weiling township of Qilihe District, Lanzhou. Typical symptoms of root and bulb rot were observed in Lanzhou lily fields. The disease incidence was estimated up to 30%. Fragments of symptomatic roots and bulbs were surface sterilized with 75% ethanol for 10 s, 2% sodium hypochlorite for 2 min, washed three times with sterilized distilled water, and then blotted dry on sterile filter paper. Fragments were placed on PDA medium and incubated at 25 ± 1°C in darkness for 5 days and 2 isolates were purified by the single-tip culture. Colonies of the fungus were white initially, and then turned light brown to brown, raised, and with entire or undulate edges. Sclerotia were brown and produced on PDA after 25 days of incubation at 25 ± 1°C in the dark. Genomic DNA from each of the two isolates was extracted, and the internal transcribed spacer (ITS) region was amplified and sequenced with the primer pair ITS5/ITS4 (White et al. 1990). The sequences of strains QLH22LD01 and QLH22LD02 were deposited in GenBank (OR710804 and OR710805). Phylogenetic analyses were performed using the Maximum Likelihood method with ITS sequences for anastomosis groups (AG) of Rhizoctonia solani. The phylogenetic tree grouped the two isolates within the R. solani AG-6 clade with high bootstrap support (100%). PCR analysis was performed with 21 primers specifically designed to detect individual anastomosis groups or anastomosis subgroups of R. solani (Carling et al., 2002; Misawa and Kurose, 2019; Misawa et al., 2020; Okubara et al., 2008). Among the 21 specific primer pairs, only AG-6 specific primer amplified the fungal DNA, indicating that the two isolates tested belonged to the R. solani AG-6. Therefore, these two strains were identified as R. solani AG-6. For pathogenicity tests, two isolates were grown individually on sterile wheat kernels at 25 ± 1°C for 14 days. Certified pathogen-free Lanzhou lily bulbs were grown in the plastic pot filled with the sterilized soil. Fifteen 2- week-old plants were inoculated by digging the soil and burying ten infested wheat kernels in the soil adjacent to the roots. Control plants were inoculated with sterile wheat kernels using the same procedure. All plants were placed in a greenhouse with a 12h/12h light/dark photoperiod at 15 to 30°C. Fifty days after inoculation, typical root and bulb rot symptoms developed on all inoculated plants, similar to symptoms observed in the field, whereas control plants remained symptomless. Pathogenicity test was performed three times with similar to symptoms observed in the field. Finally, the fungi were reisolated from the symptomatic plants and identified by molecular analysis as the isolates used for inoculation, thus fulfilling Koch's postulates. To our knowledge, this is the first confirmed report of R. solani AG-6 causing root and bulb rot on Lanzhou lily in China. Our findings improve knowledge about R. solani AGs occurring in Lanzhou lily fields in China. Due to serious damages caused by this disease in recent years in China, further studies should be conducted to investigate the diversity, prevalence, disease control measures and fungicide sensitivity of AGs distributed in the main Lanzhou lily-producing states in China.

First Confirmed Report of Rhizoctonia solani AG-7 Causing Potato Stem Canker in Mexico.

Rhizoctonia solani, is the causal agent of black scurf and stem canker of potatoes (Solanum tuberosum L.) throughout the world. In November 2021, stem canker symptoms were observed in two potato fields located in Ahome, Sinaloa, Mexico. The disease incidence was estimated up to 15%. For fungal isolation, fragments of symptomatic stems were surface sterilized with 2% sodium hypochlorite for 2 min, rinsed with sterilized distilled water, and blotted dry on sterile filter paper. Fragments were placed on PDA medium and incubated at 25°C in darkness for 4 days. Rhizoctonia-like colonies were consistently obtained and 12 isolates were purified by the hyphal-tip method. Fungal colonies on PDA were white initially and then turned brown, raised, and with entire or undulate edges. Septate hyphae were hyaline, smooth, and branched at right angles with a septum near the point of branching. Microscopic examination by staining with 1% safranin O and 3% KOH solution showed multinucleate cells. The morphological features of the isolates resembled those of Rhizoctonia solani (Sneh et al. 1991). Four representative isolates were selected for molecular analysis and pathogenicity tests. The isolates were deposited in the Culture Collection of Phytopathogenic Fungi at the Research Center for Food and Development (Culiacán, Sinaloa) under accession nos. CCLF267, CCLF274, CCLF277, and CCLF279. For molecular identification, genomic DNA from each of the four isolates was extracted, and the internal transcribed spacer (ITS) region was amplified, and sequenced with the primer pair ITS5/ITS4 (White et al. 1990). The sequences were deposited in GenBank (accession nos. OP784258 to OP784261). Phylogenetic analyses were performed using the Maximum Likelihood method with ITS sequences for anastomosis groups (AG) of Rhizoctonia solani. The phylogenetic tree grouped the four isolates within the R. solani AG-7 clade with high bootstrap support (100%). For pathogenicity tests, certified pathogen-free potato mini-tuber (cv. Fianna) were placed in a polystyrene pot (1 L) filled with a 5 cm layer of a sterile substrate composed of soil and peat moss (2:1 w/w). One rice grain (20 mg) colonized with each isolate was placed 10 mm above the uppermost sprout tip and covered with the sterile substrate (Inokuti et al. 2019). Control plants were inoculated with sterile rice grains. All pots were transferred to a greenhouse where the temperature ranged from 20 to 32°C. Stem necrosis symptoms were observed on all inoculated plants 25 days after emergence, whereas control plants remained symptomless. Pathogenicity test was performed twice with similar results. Fungi were reisolated from the infected stems and found to be morphologically identical to the isolates used for inoculation, thus fulfilling Koch's postulates. The AG-7 has been previously reported to cause potato diseases in South Africa (Truter and Wehner 2004). In Mexico, Carling et al. (1998) reported the presence of an isolate of R. solani AG-7 obtained from a potato tuber-borne sclerotium in Toluca; however, there is no information about the methodology used for the characterization of that isolate. To our knowledge, this is the first confirmed report of R. solani AG-7 causing potato stem canker in Mexico. Our findings improve knowledge about R. solani AGs occurring in potato fields in Mexico. So, further studies should be conducted to investigate the diversity, prevalence, and fungicide sensitivity of AGs distributed in the main potato-producing states in Mexico.

Assessment of Genetic Variability and Evolutionary Relationships of Rhizoctonia solani Inherent in Legume Crops.

Rhizoctonia solani is one of the most common soil-borne fungal pathogens of legume crops worldwide. We collected rDNA-ITS sequences from NCBI GenBank, and the aim of this study was to examine the genetic diversity and phylogenetic relationships of various R. solani anastomosis groups (AGs) that are commonly associated with grain legumes (such as soybean, common bean, pea, peanut, cowpea, and chickpea) and forage legumes (including alfalfa and clover). Soybean is recognized as a host for multiple AGs, with AG-1 and AG-2 being extensively investigated. This is evidenced by the higher representation of sequences associated with these AGs in the NCBI GenBank. Other AGs documented in soybean include AG-4, AG-7, AG-11, AG-5, AG-6, and AG-9. Moreover, AG-4 has been extensively studied concerning its occurrence in chickpea, pea, peanut, and alfalfa. Research on the common bean has been primarily focused on AG-2, AG-4, and AG-1. Similarly, AG-1 has been the subject of extensive investigation in clover and cowpea. Collectively, AG-1, AG-2, and AG-4 have consistently been identified and studied across these diverse legume crops. The phylogenetic analysis of R. solani isolates across different legumes indicates that the distinct clades or subclades formed by the isolates correspond to their specific anastomosis groups (AGs) and subgroups, rather than being determined by their host legume crop. Additionally, there is a high degree of sequence similarity among isolates within the same clade or subclade. Principal coordinate analysis (PCoA) further supports this finding, as isolates belonging to the same AGs and/or subgroups cluster together, irrespective of their host legume. Therefore, the observed clustering of R. solani AGs and subgroups without a direct association with the host legume crop provides additional support for the concept of AGs in understanding the genetic relationships and evolution of R. solani.

Anastomosis Grouping and Phylogenetic analysis of Rhizoctonia Isolates on Wheat in Türkiye

The aim of this study is to determine the species and to evaluate the genetic diversity of the pathogenic and nonpathogenic Rhizoctonia spp. and anastomosis groups (AG) from wheat plants and rhizosphere soils in Turkey. Rhizoctonia species were isolated from plants and rhizosphere soils in wheat fields in 5 provinces in the Central Anatolian Region of Türkiye. As a result of the isolations, a total of 88 multinucleate (MN) and binucleate (BN) Rhizoctonia isolates were obtained. Identifications of the isolates were determined by rDNA-ITS sequence analyzes. The identified isolates belonged to MN Waitea circinata var. zeae, W. circinata var. oryzae, W. circinata var. circinata, MN Rhizoctonia solani AG 2-1, 2-2, 3, 4 HGII, 4 HGIII, 5, 8, 11 and BN AG A, DI, E, G, H, I, I-like, K. The most isolated group was W. circinata var. circinata. In pathogenicity studies, the most virulent group was determined as R. solani AG 4. Among the binucleate isolates, groups other than R. cerealis AG DI were not found to be pathogenic. Neighbor-joining phylogenetic trees of isolates were constructed from rDNA-ITS sequences. As a result of this study, the regional distribution of MN and BN Rhizoctonia AG isolates in important wheat production areas in the Central Anatolia Region, Turkey was determined. In addition, this study is the first comprehensive study in which the genetic diversity of Rhizoctonia AGs isolates obtained from wheat and rhizosphere soils in the region was evaluated with a molecular approach.

Yield Loss and Integrated Disease Control of Rhizoctonia solani AG2-1 Using Seed Treatment and Sowing Rate of Oilseed Rape.

Rhizoctonia solani anastomosis group (AG) 2-1 is an ubiquitous soilborne pathogen causing severe damping-off of oilseed rape (OSR). In the absence of varietal resistance to AG2-1, there are limited methods for integrated disease management. The objectives of these field studies were to quantify yield losses due to AG2-1 and to determine the effectiveness of integrated control using sedaxane, fludioxonil, and metalaxyl-M applied as seed treatment on two OSR genotypes at a sowing rate of 40 (low) or 80 (high) seeds m-2. Crop assessments of green area index (GAI), vigor, and cabbage stem flea beetle (CSFB) Psylliodes chrysocephala damage were carried out at GS16, while pathogen DNA in soil was quantified using real-time PCR at GS32. Yield and seed weight losses of 41 and 18%, respectively, were associated with reduced establishment, GAI, vigor, and delayed development and flowering of OSR. Seed treatment reduced AG2-1 DNA in soil by 80%, resulting in a 94, 16, and 64% increase of establishment, thousand seed weight (TSW), and yield, respectively. Seed treatment also mitigated the effects of AG2-1 on delaying plant development, resulting in increased uniformity of crop flowering. OSR plants infected with AG2-1 suffered 27% more damage by the CSFB, indicating positive pathogen-pest interaction at the expense of the OSR host. Optimum control of AG2-1 infection was achieved by integrating low sowing rate and seed treatment. However, under dual pest and pathogen attack, high sowing rates should be combined with the use of seed treatment to mitigate seedling death and delayed development caused by AG2-1 and CSFB damage.

Heritability and gene functions associated with sclerotia formation of Rhizoctonia solani AG-7 using whole genome sequencing and genome-wide association study.

Sclerotia are specialized fungal structures formed by pigmented and aggregated hyphae, which can survive under unfavourable environmental conditions and serve as the primary inocula for several phytopathogenic fungi including Rhizoctonia solani. Among 154 R. solani anastomosis group 7 (AG-7) isolates collected in fields, the sclerotia-forming capability regarding sclerotia number and sclerotia size varied in the fungal population, but the genetic makeup of these phenotypes remained unclear. As limited studies have focused on the genomics of R. solani AG-7 and the population genetics of sclerotia formation, this study completed the whole genome sequencing and gene prediction of R. solani AG-7 using the Oxford NanoPore and Illumina RNA sequencing. Meanwhile, a high-throughput image-based method was established to quantify the sclerotia-forming capability, and the phenotypic correlation between sclerotia number and sclerotia size was low. A genome-wide association study identified three and five significant SNPs associated with sclerotia number and size in distinct genomic regions, respectively. Of these significant SNPs, two and four showed significant differences in the phenotypic mean separation for sclerotia number and sclerotia size, respectively. Gene ontology enrichment analysis focusing on the linkage disequilibrium blocks of significant SNPs identified more categories related to oxidative stress for sclerotia number, and more categories related to cell development, signalling and metabolism for sclerotia size. These results indicated that different genetic mechanisms may underlie these two phenotypes. Moreover, the heritability of sclerotia number and sclerotia size were estimated for the first time to be 0.92 and 0.31, respectively. This study provides new insights into the heritability and gene functions related to the development of sclerotia number and sclerotia size, which could provide additional knowledge to reduce fungal residues in fields and achieve sustainable disease management.

Population Genetics and Anastomosis Group's Geographical Distribution of Rhizoctonia solani Associated with Soybean.

Rhizoctonia solani is a species complex composed of many genetically diverse anastomosis groups (AG) and their subgroups. It causes economically important diseases of soybean worldwide. However, the global genetic diversity and distribution of R. solani AG associated with soybean are unknown to date. In this study, the global genetic diversity and distribution of AG associated with soybean were investigated based on rDNA-ITS sequences deposited in GenBank and published literature. The most prevalent AG, was AG-1 (40%), followed by AG-2 (19.13%), AG-4 (11.30%), AG-7 (10.43%), AG-11 (8.70%), AG-3 (5.22%) and AG-5 (3.48%). Most of the AG were reported from the USA and Brazil. Sequence analysis of internal transcribed spacers of ribosomal DNA separated AG associated with soybean into two distinct clades. Clade I corresponded to distinct subclades containing AG-2, AG-3, AG-5, AG-7 and AG-11. Clade II corresponded to subclades of AG-1 subgroups. Furthermore, AG and/or AG subgroups were in close proximity without corresponding to their geographical origin. Moreover, AG or AG subgroups within clade or subclades shared higher percentages of sequence similarities. The principal coordinate analysis also supported the phylogenetic and genetic diversity analyses. In conclusion, AG-1, AG-2, and AG-4 were the most prevalent AG in soybean. The clade or subclades corresponded to AG or AG subgroups and did not correspond to the AG's geographical origin. The information on global genetic diversity and distribution will be helpful if novel management measures are to be developed against soybean diseases caused by R. solani.

Diversity and Aggressiveness of Rhizoctonia spp. from Nebraska on Soybean and Cross-Pathogenicity to Corn and Wheat.

Rhizoctonia and Rhizoctonia-like species of fungi that cause disease are known to have varying host ranges and aggressiveness. Accurate identification of these species causing disease is important for soybean disease management that relies upon crop rotation. The anamorphic genus Rhizoctonia contains several diverse species and anastomosis groups (AGs) including some known soybean pathogens, such as Rhizoctonia solani, whereas for others the ability to cause disease on soybean has not been well described. The present study was conducted to identify the predominant species and AG of Rhizoctonia from soybean, corn, and wheat fields that are pathogenic on soybean and characterize cross-pathogenicity to common rotational crops, corn and wheat. We surveyed for Rhizoctonia spp. in Nebraska; isolates were identified to species and AG, and aggressiveness was assessed. A total of 59 R.zeae isolates, 49 R.solani, nine binucleate Rhizoctonia, three R.circinata, and two R.oryzae isolates were collected in 2016 and 2017 from a total of 29 fields in 15 counties. The most abundant R.solani AGs were AG-4, AG-1 IB, AG-2-1, AG-3, and AG-5. R.solani AG-4 and R. zeae were found in all three regions of the state (west, central, and eastern). Some isolates that were most aggressive to soybean seedlings were cross-pathogenic on both wheat and corn. In addition, R. zeae was pathogenic on soybean when evaluated at 25°C, which is warmer than temperatures used previously, and isolates were identified that were aggressive on soybean and cross-pathogenic on both corn and wheat.

Establishment and Application of a Multiplex PCR Assay for the Rapid Detection of Rhizoctonia solani Anastomosis Group (AG)-3PT, the Pathogen Causing Potato Black Scurf and Stem Canker.

Rhizoctonia solani anastomosis group 3 (AG-3) is the main causative agent of the soil-borne disease known as potato black scurf, which poses a huge threat to potato production. Rapid and accurate identification of R. solani AG-3 isolates in soil and potato seed tubers prior to planting is essential for good production. In this study, a multiplex PCR assay was established for the detection of R. solani AG-3. Two pairs of target-specific primers were designed from sequences for endopolygalacturonase and pyridoxine biosynthesis genes downloaded from GenBank. The main factors influencing PCR amplification, such as annealing temperature and primer concentration, were optimized. Results show that the proposed multiplex PCR assay is highly sensitive and specific for the target genes in the pathogen even when the DNA concentration is reduced to 20 fg/μL. The resulting calibration plot shows a linear relationship between electrophoretic band peaks and genomic DNA concentration (R2 = 0.98). The primer specificity was confirmed by applying them to other R. solani AG groups and plant pathogen species on which no amplicons were produced. Using the primers, we successfully detected small amounts of R. solani AG-3 present in soil and potato tuber samples. Taken together, the detection assay developed in this study has high sensitivity, strong specificity, and accuracy and can be used to detect and identify soil and potato seed tubers infected with Rhizoctonia solani AG-3.

Molecular identification and pathogenicity of Rhizoctonia solani and Pythium spp. associated with damping‐off disease on baby leafy vegetables in Greece

AbstractIn the last years, leafy vegetables cultivated as baby leaves have been established in the market and have attracted the interest of consumers throughout the world. During the growing seasons of 2019 and 2020, 97 isolates of Rhizoctonia solani and 112 isolates of Pythium spp. were obtained from baby leaf vegetables exhibited damping‐off symptoms. Representative isolates of R. solani from each surveyed plant species were characterized using sequence analysis of the internal transcribed spacer (rDNA‐ITS) region. Isolates were identified as belonging to four anastomosis groups (AGs): AG2‐1, AG‐IB, AG4‐HGI and AG4‐HGIII. AG4‐HGI was the most prevalent group and phylogenetic analysis showed that the isolates were distinctly separated according to their AGs. Pathogenicity among the four AGs on 23 plant species varied considerably, from not susceptible to highly susceptible, while, in general, AGs did not exhibit host specificity. Furthermore, a total of 112 Pythium spp. isolates were obtained. The ITS region and the cytochrome oxidase II (coxII) gene were amplified, and three Pythium spp. were identified (P. ultimum, P. aphanidermatum and P. sylvaticum), which were used further for maximum‐likelihood phylogenetic analysis. The pathogenicity of representative isolates was assessed in vitro and in vivo on 10 plant species. In general, all three tested Pythium spp. were virulent when used in vitro, while P. ultimum was the most virulent in vivo. This is the first comprehensive study aimed at determining the occurrence of specific R. solani AGs and Pythium spp. derived from baby leafy vegetables exhibiting damping‐off symptoms in Greece.

First Report of Black Scurf Caused by Rhizoctonia solani AG 2-2IV on Potato tubers in Heilongjiang Province, China.

Black scurf and stem canker on potato (Solanum tuberosum L.), caused by Rhizoctonia solani, is one of the most important soil-borne diseases throughout the world. Isolates of R. solani anastomosis group (AG) 3-PT have been reported as the predominant cause of the disease on potato (Carling 1996) and the same results were also obtained in Heilongjiang Province, China (Yang et al. 2017). In October 2020, 14 diseased potato tubers (cv. Youjin-885) with symptoms typically associated with black scurf were found in Hegang City of Heilongjiang in Northeast China, where potatoes are grown for propagation in the breeding nursery. Pieces of sclerotia were removed from the surface of the potato and were surface sterilized with 70% ethanol for 30 s and 0.5% NaClO for 1 min, then rinsed three times with sterile distilled water and placed on potato dextrose agar (PDA) at 25°C in the dark. After incubation for 48 to 72 h, mycelia resembling Rhizoctonia were microscopically examined for morphological characteristics, and hyphal tips transferred to fresh plates of PDA. The characteristics of the observed isolate were typical of R. solani Kühn, which include hyphal branching at right angles, a septum near the branching point and a slight constriction at the branch base (Yang et al. 2015). Hyphal cells were also determined to be multinucleate by staining with 1% safranin O and 3% KOH solution (Bandoni 1979). PCR amplification and DNA sequencing of the internal transcribed spacer (ITS) region of the ribosomal DNA (rDNA) was performed by using the universal primers ITS4/ITS5 (White et al. 1990). The resulting sequence of 700 bp (GenBank accession no. OL770460) showed more than 99% identity to AG 2-2IV isolates present in GenBank (e.g. AB911322; KR259910). On the basis of morphological characteristics and molecular analysis, the isolate was identified as R. solani AG 2-2IV. Pathogenicity of the isolate was tested in greenhouse conditions. Pathogen-free minitubers (cv. Favorita) of approximately the same size (10 to 20 g) were allowed to sprout at room temperature for 10 days. The minitubers were then planted in autoclaved soil in a plastic pot (4 L capacity), placed in a greenhouse at 18 - 27°C (night-day) with 50% relative humidity and watered as required. The pots were inoculated with 7-mm-diameter mycelial plugs (from one PDA petri plate) near the minituber, which was then covered with potting mix. The control pots were inoculated with sterile plugs of PDA. Each treatment consisted of 10 plants, and the experiment was repeated three times. Two months after stems emerged, plants and progeny tubers were harvested and assessed for disease. Stem cankers typical of R. solani infection and black scurf were observed on plants grown in pots inoculated the mycelial plugs, but the control plants remained disease free. Fungi reisolated from symptomatic stems and tubers were identified as R. solani AG 2-2IV using morphological characters and ITS sequences.Sclerotia were observed on PDA by incubating at 25oC in the dark. Although eight AGs have been previously shown to cause black scurf and stem canker in Heilongjiang (Li et al. 2014; Yang et al. 2015; Yang et al. 2017; Yang et al. 2019; Yang et al. 2020), to our knowledge, this is the first report of AG 2-2IV causing disease on potatoes in Heilongjiang Province, the main potato seed production area of China. Early detection of R. solani AG 2-2IV during potato seed production is necessary to prevent its dispersal via infected tubers to other fields across China. The information of which AG is present will assist in developing management strategies for this disease.

First Report of Collar and Root Rot of Faba Bean Caused by Rhizoctonia solani AG-2-2 IIIB in Bangladesh.

Faba bean (Vicia faba L.) is an underutilized promising grain legume commercially grown in central and northern part of Bangladesh (Yasmin et al. 2020). In January 2021, faba bean plants exhibiting symptoms of collar and root rot and yellowing of leaves were observed in thirty plots of an experimental field at the Bangladesh Agricultural University (24.75° N, 90.50° E), Mymensingh, Bangladesh. Infected plants had dark brown to black lesions on the roots, extending above the collar region. An average disease incidence and severity was 7.16% and 6.91%, respectively. Eight diseased plants were collected from the field by uprooting one plant from each of eight randomly selected experimental plots and surface disinfected with sodium hypochlorite (0.2%) for 3 min followed by 1 min in ethanol (70%), and then rinsed three times with distilled water and dried on sterile paper towels. Collar and root pieces (5×5 mm) of symptomatic tissues were placed on Potato Dextrose Agar (PDA). Plates were incubated at 25°C for three days and isolates were purified from single-tip culture. The isolates produced brown colored mycelia often with brown sclerotia. Under microscope, fungal colonies exhibited right-angled branching with constriction at the base of hyphal branches and a septum near the originating point of hyphal branch consistent with the description of Rhizoctonia solani Kuhn (Sneh et al. 1991). The isolates grew at 35°C on PDA (5 mm/24). Molecular identification of the isolates BTRFB1 and BTRFB7 was determined by sequencing the rDNA internal transcribed spacer (ITS) region using primers ITS1 and ITS4 (White et al. 1990). A BLAST search showed that the sequences (GenBank Accession nos. MZ158299.1 and MZ158298.1) had 99.28% similarity with R. solani isolates Y1063 and SX-RSD1 (GenBank Accession nos. JX913811.1 and KC413984.1, respectively). Phylogenetic analysis revealed that the present isolates grouped with R. solani anastomosis group AG-2-2 IIIB. To confirm pathogenicity, both isolates were grown individually on sterile wheat kernels at 28°C for 6 days (D'aes et al. 2011). Faba bean seedlings were grown in plastic pots containing sterile potting mix (field soil/composted manure/sand 2:2:1 [v/v]). Two-week-old plants were inoculated by placing five infested wheat seeds adjacent to the roots. Control pots were inoculated with sterile wheat kernels using the same procedure. Plants were placed in a growth room with a 16 h/8 h light/dark photoperiod at 25 ± 2°C after inoculation. Fifteen days after inoculation, typical collar and root rot symptoms were developed on inoculated plants, similar to symptoms observed in the field. Control plants remained non-symptomatic. Finally, six isolates of R. solani were isolated from the symptomatic plants and identified by morphological and molecular analysis. Rhizoctonia solani is the causal agent of seed and root rot, hypocotyl canker, and seedling damping-off diseases of faba bean in many other countries (Rashid and Bernier 1993; Assunção et al. 2011). To our knowledge, this is the first confirmed report of Rhizoctonia solani causing collar and root rot of faba bean in Bangladesh. This finding will be helpful for the development of management strategies to control this disease and to expand the production of faba bean in Bangladesh.

First Report of Soybean Seedling Disease caused by Rhizoctonia solani AG-7 in Taiwan.

From August to November 2020, reduced emergence and damping-off of soybean seedlings were observed in two fields (Benzhou and Wandan) in Taiwan. Disease incidence was approximately 40% in Benzhou by field scouting. The roots of damping-off seedlings were brown. Affected seedlings could be easily pulled out from the soil and the lesions on the roots/stem were generally dry and sunken. These symptoms suggested the possibility of Rhizoctonia infection. Soil surrounding symptomatic seedlings were collected to bait the potential pathogen and symptomatic plants were used for pathogen isolation. The diseased tissues were washed with tap water and surface-disinfected with 1% bleach before placing on the Dexon selection medium at 26°C for 2 days (Ko and Hora 1971). Hyphae were transferred to potato dextrose agar (PDA), and a brown colony with brown and irregular-shaped sclerotia grew from 90 out of 99 isolates. The hyphae exhibited typical characteristics of Rhizoctonia solani, including a constriction and a septum near the end of branching hyphae (Ajayi-Oyetunde and Bradely, 2018). Two isolates from Benzhou and two isolates from Wandan were tested for their pathogenicity, and eight surface-disinfected seeds were distributed evenly on the water agar plates covered by 2-day-old mycelia at 25°C in dark for 7 days. All isolates caused cotyledon rot and reduced germination. To verify their pathogenicity in pots, double-sterilized sorghum seeds were inoculated with two strains and incubated at 25°C for 2 weeks to be used as fungal inoculum (Ajayi-Oyetunde and Bradely, 2017). A layer of 15 ml of fungal inoculum was placed 5 cm beneath the soil surface in pots. Four soybean seeds were planted approximately 3 cm above the inoculum in each pot. After two weeks, reddish lesions on the hypocotyls or taproots of all seedlings in the inoculated pots were observed, while seedlings in the control pots inoculated with sterile sorghum seeds remained healthy. The pathogen was re-isolated from lesions and had identical morphology to the original isolates. To characterize the fungal identity, the internal transcribed spacer (ITS) was sequenced using the primers ITS1/ITS4 (Sharon et al., 2006). Using BLASTN in the NCBI database, the sequence (GenBank no. MW410857 and MW410858) showed 100% (639/639 bp) similarity to KF907734 and 99.83% (635/636 bp) similarity to AF354099, both belong to R. solani anastomosis group 7 (AG-7) (Hua et al. 2014; Gonzalez et al. 2001). Phylogenetic analysis comparing sequences with different AGs (Ajayi-Oyetunde and Bradely, 2017) grouped our isolates within the AG-7 clade with a 100% bootstrap confidence. In the anastomosis test, an incompatible zonation and unequal mycelial growth rates were observed when AG-7 isolates were paired with an AG-1 IA isolate. On the other hand, the compatible tuft reaction was observed when two AG-7 isolates were paired, and the compatible merge reaction was observed in the self-pairing tests (Macnish et al. 1997). Accordingly, the molecular and morphological characterizations confirmed the causal pathogen as R. solani AG-7. R. solani AG-7 was first reported on radishes in Japan (Homma et al., 1983), first found on carnation in Taiwan (Lo et al., 1990), and in field soils of various crops but not soybean (Chuang, 1997). It was suggested that Rhizoctonia diseases of soybean may be present in Taiwan, but molecular confirmation was lacking (Anonymus, 1979). As R. solani AG-7 causes diseases of soybean in the US and Japan (Baird et al., 1996), the importance of AG-7 as an endemic pathogen of soybean in Taiwan should be recognized and its prevalence determined as a first step to managing this disease.

Sensitivity of Rhizoctonia solani Anastomosis Group 2-2 Isolates from Soybean and Sugar Beet to Selected SDHI and QoI Fungicides.

Rhizoctonia solani causes root and stem diseases on soybean and sugar beet, and fungicides are commonly used to manage these diseases. Quinone outside inhibitor (QoI) fungicides (pyraclostrobin and azoxystrobin) have been used for in-furrow and postemergence application since 2000. Succinate dehydrogenase inhibitor (SDHI) fungicides (sedaxane, penthiopyrad, and fluxapyroxad) became popular seed treatments after their registration in Minnesota and North Dakota between 2012 and 2016. Periodic monitoring of sensitivity to these fungicides in R. solani anastomosis group (AG) 2-2 is important to detect potential shifts in sensitivity over time. R. solani AG 2-2 isolates (n = 35) collected from soybean and sugar beet in Minnesota and North Dakota were evaluated invitro for sensitivity. Isolates were considered as baseline or nonbaseline for the above-mentioned fungicides based on previous potential exposure. The effective concentration (EC50) required to suppress radial fungal growth by 50% was determined. The mean EC50 values for sedaxane, penthiopyrad, fluxapyroxad, and pyraclostrobin were 0.1, 0.15, 0.16, and 0.25 (µg ml-1), respectively. The mean EC50 value for azoxystrobin for 22 isolates was 0.76 to 1.56 µg ml-1; and EC50 could not be determined for 13 isolates because of <50% inhibition at the highest concentrations used. The EC50 values for the QoI fungicides did not differ significantly between baseline and nonbaseline isolates. EC50 values for SDHI fungicides were significantly higher for isolates collected from soybean than from sugar beet, and isolates collected from both crops had similar EC50 values for pyraclostrobin. All SDHI fungicides and pyraclostrobin effectively suppressed R. solani isolates from soybean and sugar beet at low concentrations invitro.

Effect of Low Temperature on the Aggressiveness of Rhizoctonia solani AG 2-2 Isolates on Sugar Beet (Beta vulgaris) Seedlings.

Rhizoctonia solani anastomosis group (AG) 2-2 can cause seedling damping-off in sugar beets and substantial losses may occur in all regions where beets are grown. Sugar beets are planted early in the season when soil temperatures are low in order to maximize the length of the growing season and minimize the risk of damping-off. However, predictive models that indicate there is little to no risk of Rhizoctonia damping-off at temperatures <15°C may not be entirely reliable. We tested this possibility by inoculating sugar beet seedlings in a growth chamber at 11°C with 35 R. solani AG 2-2 isolates that were representative of the genetic diversity present in AG 2-2. Although disease progress and growth rate were greatly reduced at 11°C, considerable disease symptoms did develop in inoculated plants. Three weeks after inoculation, 16% of the plants were dead and 77% of the isolates tested had average disease severity scores that were significantly greater than those of the mock inoculated control. This confirms our concern about the possibility for low-temperature infection of sugar beets and indicates that waiting until the soil warms up to above 15°C to apply fungicide could leave the crop at risk. Aggressiveness does not appear to be related to subgroup or growth rate but rather depends on the response of the specific isolate to low temperature.

Yield Losses and Control by Sedaxane and Fludioxonil of Soilborne Rhizoctonia, Microdochium, and Fusarium Species in Winter Wheat.

Soilborne Rhizoctonia, Microdochium, and Fusarium species are major causal agents of seedling and stem-base diseases of wheat. Currently, seed treatments are considered the most effective solution for their control. Rhizoctonia solani anastomosis groups (AGs) 2-1 and 5, R. cerealis, Microdochium, and Fusarium spp., were used in series of field experiments to determine their capability to cause soilborne and stem-base disease and to quantify their comparative losses in the establishment and yield of wheat. The effectiveness and response to seed treatment formulated with 10 g sedaxane and 5 g fludioxonil 100 kg-1 against these soilborne pathogens were also determined. Our results showed that damping-off caused by soilborne R. cerealis was associated with significant reductions in the emergence and establishment, resulting in stunted growth and low plant numbers. The pathogen also caused sharp eyespot associated with reductions in the ear partitioning index. R. solani AG 2-1 and AG 5 were weakly pathogenic and failed to cause significant damping-off, root rot, and stem-base disease in wheat. Fusarium graminearum and F. culmorum applied as soilborne inoculum failed to cause severe disease. Microdochium spp. caused brown foot rot disease and soilborne M. nivale reduced wheat emergence. Applications of sedaxane and fludioxonil increased plant emergence and reduced damping-off, early stem-base disease, and brown foot rot, thus providing protection against multiple soilborne pathogens. R. cerealis reduced the thousand grain weight by 3.6%, whereas seed treatment including fludioxonil and sedaxane against soilborne R. cerealis or M. nivale resulted in a 4% yield increase.

Identification and expression analysis of pathogenicity-related genes of Rhizoctonia solani anastomosis groups infecting rice.

The online version contains supplementary material available at 10.1007/s13205-021-02934-1.