Solani AG Research Articles

Assessing Biofungicides and Host Resistance against Rhizoctonia Large Patch in Zoysiagrass.

Rhizoctonia large patch (Rhizoctonia solani AG2-2 LP) significantly reduces turfgrass quality, aesthetics, and playability. Synthetic fungicides are commonly used for managing this disease, but they present high costs, potential for fungicide resistance, and environmental concerns. We conducted in vitro assays to test the effectiveness of three biofungicides, seven synthetic fungicides, and ten combinations against R. solani. We then assessed seven spray programs that included Bacillus subtilis QST713 and propiconazole, either alone or tank-mixed, on zoysiagrass 'El Toro' in a growth chamber and in field trials. Biofungicide B. subtilis QST713 reduced pathogen growth by up to 100% in vitro. B. subtilis QST713 alone or combined with synthetic fungicides and/or in rotation was as effective as the standalone synthetic fungicide, reducing disease severity and AUDPC by 81 and 77% (growth chamber) and by 71 and 52% (field), respectively, while maintaining acceptable turfgrass quality. Additionally, we screened zoysiagrass genotypes and advanced breeding lines against three R. solani isolates in growth chamber studies. Five genotypes and two breeding lines demonstrated resistance to Rhizoctonia large patch across isolates, highlighting their potential for developing disease-resistant cultivars. Our findings suggest that integrating biofungicides, resistant cultivars with chemical controls offer sustainable and effective strategies for managing Rhizoctonia large patch.

First Report of Rhizoctonia solani AG 4 HG-III Causing Stem Lesion of Watermelon in Idaho.

In July 2022, stem lesions, approximately 4 to 5 cm in length as well as leaf wilt and dark brown necrosis on stems and roots were observed in two fields in Southwest Idaho on 20 to 30% of watermelons (Citrullus lanatus). To determine the causal agent, isolations were attempted from symptomatic tissue. The surface of the affected material was disinfected with 0.6% sodium hypochlorite for 1 min and rinsed three times with sterile water. Approximately 2 mm3 sections of tissue were plated on water agar amended with 0.02% penicillin and 0.08% streptomycin and incubated at room temperature for 7 days. Fungal colonies were tentatively identified as Rhizoctonia from right-angle branching and septate hyphal structures, slight constriction and septum near the branch base, and the production of 1 to 2 mm white to light brown irregularly shaped sclerotia. Single hyphal tips were transferred to potato dextrose agar (PDA) and grown at room temperature. Approximately ten isolates from each field with a consistent macromorphology were observed. These isolates had light brown mycelia, produced sclerotia at ambient temperature with no exposure to continuous light, and a representative isolate, designated D22-110 was selected for sequencing and pathogenicity testing. For isolate D22-110, mycelia were removed with a scalpel after 7 days of growth, for DNA extraction and sequencing of the rDNA internal transcribed spacer (ITS) region as previously described (White et al., 1990). A 726 bp product was generated and the sequence was submitted to GenBank (Accession No. OQ794049). NCBI-BLAST indicated this sequence was 99% identical (631 of 634 bp and 632 of 634 bp identical) with known reference isolates previously identified as R. solani AG 4 HG-III (Accession No. AF354075 and AF354076, respectively) from a phylogenetic study (Gonzalez et al., 2001). Pathogenicity testing was performed twice on two-week-old seedlings of watermelon cultivars Endless Summer and Wingman in greenhouse conditions (29oC, 12 h daylight). Two disks (3 mm diam) from 7-day-old plates of PDA were placed around each seedling at the root and stem convergence point. Ten seedlings were mock-inoculated with sterile PDA plugs as a control. Approximately 35% damping-off incidence was observed on inoculated seedlings six days post-inoculation, while control seedlings remained healthy. At 20 days post-inoculation, 20 (first trial) and 34 seedlings (second trial) were assessed for visible stem and root lesions. Incidence of stem lesions occurred on 90% of seedlings, with 80% of seedlings possessing lesions greater than 10 mm in diameter. Seedlings without R. solani inoculation were free of stem and root lesions. R. solani was re-isolated from symptomatic tissue, with 40% frequency of isolation, identified by right-angle branching of the hyphae thus confirming Koch's postulates. R. solani AG 4 has been reported in watermelon in the US since 1994 (Hall and Summer, 1994) but the AG 4 subgroup was not reported. AG 4 HG-III was reported in melon seedlings causing damping-off in Kyrgyzstan (Erper et al. 2016). In other hosts, AG 4 HG-III was found in potatoes in South Africa (Muzhinji et al., 2014), buckwheat and foxtail millet in China (Zhou et al., 2015; Hao et al., 2023), broccoli and spinach (Kuramae et al., 2003) and turnip green (Sekiguchi et al., 2015). To the best of our knowledge, this is the first report of R. solani AG 4 HG-III causing disease in watermelon in Idaho. Given the the rate of disease incidence observed in the field, growers should consider avoiding planting alternative host crops to minimize inoculum buildup.

First Report of Rhizoctonia solani AG1-IB Causing Tobacco Target Spot in China.

Tobacco target spot, caused by Rhizoctonia solani Kühn, induces shot-hole lesions on leaves that that significantly reduce yield and quality of tobacco. In July 2022, samples (n=5) with target spot were collected from three tobacco fields, one each in Puer (22.63°N, 100.72°E, cv. Yunyan87) and Mengzi (23.26°N, 103.36°E, cv. Yunyan87) of Yunnan province and one in Dandong (40.63°N, 124.18°E, cv. Liaoyan17) of Liaoning province, China; disease incidence in these fields was approximately 30%~40%. Initial symptoms (2- to 3-mm-diameter lesions) appeared on the middle to lower leaves, then expanded to 2 to 3 cm in diameter and developed the shot-hole appearance. Pieces of tissue (5×5 mm) were cut from the edge of lesions, surface sterilized, rinsed in sterile water, then placed on the surface of water agar (WA) and incubated at 25℃ for 2 days in the dark. Single hyphal tips were taken from fungal isolates identified as R. solani based on the morphological traits (Tsror 2010), then transferred onto potato dextrose agar (PDA) and cultured for 3 d as described above. A total of 15 pure cultures were obtained. With the exception of YN-3 (isolated from Puer), YN-62 (isolated from Mengzi) and LN-95(isolated from Dandong) strains, which exhibited hyphal fusion reaction with AG1-IB standard strain, all the other strains demonstrated hyphal fusion with AG-3 standard strain (Ogoshi 1987). Genomic DNA of these three strains were extracted by the CTAB method and ITS regions of rDNA were sequenced (White et al. 1990). The sequences were deposited in GenBank with accession No. OR770079, OR770080 and OR770082. All the three rDNA-ITS sequences exhibited 99.85% similar to AG1-IB found in GenBank, and a phylogenetic tree using a neighbor-joining method grouped the three strains within the R. solani AG-1 IB clade. Therefore, based on the hyphal fusion reaction and molecular methods, these isolates were identified as R. solani AG1-IB. To determine pathogenicity of the isolates, the healthy leaves of tobacco plants (cv. Yunyan 87) were used. Five-mm-diameter mycelial plugs of the strain on PDA were inoculated on leaves that had been previously wounded with a sterile needle, and cotton balls moistened with sterile water were used for moisturizing the inoculation sites. Ten leaves were inoculated for each strain and leaves inoculated with PDA plugs were as control. The experiment was conducted twice. All plants were incubated for 2 d at 15℃ to 25℃ and 90% relative humidity with a 12 h photoperiod/day. Irregularly shaped lesions appeared on the leaves around each of the inoculated sites, but not on control leaves. The pathogens were reisolated and confirmed be R. solani AG1-IB by hyphal fusion and molecular identification tests as previously described, thereby fulfilling Koch's postulates. It has been reported that AG-3, AG-2 (Mercado Cardenas et al. 2012), AG-5 (Wang et al. 2023) and AG-6 (Sun et al. 2022) of R. solani could cause tobacco target spot, but AG-3 is considered the main causal agent (Marleny Gonzalez et al. 2011). To our knowledge, this is the first report of AG1-IB causing tobacco target spot in China and worldwide. The AG1-IB strain has a wide host range including cabbage, mint, lettuce, beans, and rice (Gonzalez et al. 2006). The discovery poses a new challenge for the prevention and control of tobacco target spot, especially when contemplating disease management strategies such as crop rotation and fungicide treatments.

Biological characteristics and metabolic phenotypes of different anastomosis groups of Rhizoctonia solani strains

BackgroundRhizoctonia solani is an important plant pathogen worldwide, and causes serious tobacco target spot in tobacco in the last five years. This research studied the biological characteristics of four different anastomosis groups strains (AG-3, AG-5, AG-6, AG-1-IB) of R. solani from tobacco. Using metabolic phenotype technology analyzed the metabolic phenotype differences of these strains.ResultsThe results showed that the suitable temperature for mycelial growth of four anastomosis group strains were from 20 to 30oC, and for sclerotia formation were from 20 to 25oC. Under different lighting conditions, R. solani AG-6 strains produced the most sclerotium, followed by R. solani AG-3, R. solani AG-5 and R. solani AG-1-IB. All strains had strong oligotrophic survivability, and can grow on water agar medium without any nitrutions. They exhibited three types of sclerotia distribution form, including dispersed type (R. solani AG-5 and AG-6), peripheral type (R. solani AG-1-IB), and central type (R. solani AG-3). They all presented different pathogenicities in tobacco leaves, with the most virulent was noted by R. solani AG-6, followed by R. solani AG-5 and AG-1-IB, finally was R. solani AG-3. R. solani AG-1-IB strains firstly present symptom after inoculation. Metabolic fingerprints of four anastomosis groups were different to each other. R. solani AG-3, AG-6, AG-5 and AG-1-IB strains efficiently metabolized 88, 94, 71 and 92 carbon substrates, respectively. Nitrogen substrates of amino acids and peptides were the significant utilization patterns for R. solani AG-3. R. solani AG-3 and AG-6 showed a large range of adaptabilities and were still able to metabolize substrates in the presence of the osmolytes, including up to 8% sodium lactate. Four anastomosis groups all showed active metabolism in environments with pH values from 4 to 6 and exhibited decarboxylase activities.ConclusionsThe biological characteristics of different anastomosis group strains varies, and there were significant differences in the metabolic phenotype characteristics of different anastomosis group strains towards carbon source, nitrogen source, pH, and osmotic pressure.

Biogenic Synthesis of Silver and Iron Oxide Nanoparticles Using Aronia prunifolia Leaf Extract and Its Inhibitory Action Against Pathogenic Fungi

Because of the environment and abundant renewable resources, exploiting plant extracts to form metallic nanoparticles has become a promising alternative to chemical and physical methods. Numerous studies have shown that nanoparticles of silver (AgNPs) and iron oxide (Fe2O3NPs) have inhibitory effects against pathogenic fungi. In this study, we used the leaf extract of Aronia prunifolia to generate biogenic AgNPs and FeNPs, aiming to demonstrate the impact of nanoparticles on pathogenic fungi. Nanoparticles are characterized by UV-Vis, X-ray diffraction, EDX spectrum, and SEM techniques. Leaf extracts used for nanosynthesis yielded silver and iron oxide nanoparticles with distinct color changes and absorption peaks, showcasing tetragonal, pentagonal, and hexagonal shapes (15-50 nm) for silver and spherical morphology (16-60 nm) for iron oxide. The antifungal effectiveness of nanoparticles against Aspergillus fumigatus, Rhizoctonia solani Ag4 HgII, and Aspergillus flavus was investigated using a well diffusion method. Inhibition zones, ranging from 12.5 to 35.0 mm for AgNPs and 7.1 to 17.1 mm for FeNPs at concentrations of 10 to 30 µg/ml respectively, demonstrated the superior inhibitory potential of AgNPs over FeNPs. This study aims to address a gap in the literature by examining the inhibitory effects of green AgNPs and FeNPs on pathogenic fungi. Encased nanoparticles can be very useful in treating fungal infections; this will be the first investigation into the production of nanoparticles from A. prunifolia leaves.

Unveiling the structural properties and induced resistance activity in rice of Chitin/Chitosan-Glucan Complex of Rhizoctonia solani AG1 IA inner cell wall

Phytopathogen cell wall polysaccharides have important physiological functions. In this study, we isolated and characterized the alkali-insoluble residue on the inner layers of the Rhizoctonia solani AG1 IA cell wall (RsCW-AIR). Through chemical composition and structural analysis, RsCW-AIR was mainly identified as a complex of chitin/chitosan and glucan (ChCsGC), with glucose and glucosamine were present in a molar ratio of 2.7:1.0. The predominant glycosidic bond linkage of glucan in ChCsGC was β-1,3-linked Glcp, both the α and β-polymorphic forms of chitin were presented in it by IR, XRD, and solid-state NMR, and the ChCsGC exhibited a degree of deacetylation measuring 67.08 %. RsCW-AIR pretreatment effectively reduced the incidence of rice sheath blight, and its induced resistance activity in rice was evaluated, such as inducing a reactive oxygen species (ROS) burst, leading to the accumulation of salicylic acid (SA) and the up-regulation of SA-related gene expression. The recognition of RsCW-AIR in rice is partially dependent on CERK1.

Genome sequencing and comparative genome analysis of Rhizoctonia solani AG-3.

Rhizoctonia solani AG-3 is a plant pathogenic fungus that belongs to the group of multinucleate Rhizoctonia. According to its internal transcribed spacer (ITS) cluster analysis and host range, it is divided into TB, PT, and TM subgroups. AG-3 TB mainly causes tobacco target spots, AG-3 PT mainly causes potato black scurf, and AG-3 TM mainly causes tomato leaf blight. In our previous study, we found that all 36 tobacco target spot strains isolated from Yunnan (Southwest China) were classified into AG-3 TB subgroup, while only two of the six tobacco target spot strains isolated from Liaoning (Northeast China) were classified into AG-3 TB subgroup, and the remaining four strains were classified into AG-3 TM subgroup, which had a unique taxonomic status, and there was no previous report on the whole genome information of AG-3 TM subgroup. In this study, the whole genomes of R. solani AG-3 strains 3T-1 (AG-3 TM isolated from Liaoning) and MJ-102 (AG-3 TB isolated from Yunnan) isolated from tobacco target spot in Liaoning and Yunnan were sequenced by IIumina and PacBio sequencing platforms. Comparative genomic analysis was performed with the previously reported AG-3 PT strain Rhs1AP, revealing their differences in genomes and virulence factors. The results indicated that the genome size of 3T-1 was 42,103,597 bp with 11,290 coding genes and 49.74% GC content, and the genome size of MJ-102 was 41,908,281 bp with 10,592 coding genes and 48.91% GC content. Through comparative genomic analysis with the previously reported strain Rhs1AP (AG-3 PT), it was found that the GC content between the genomes was similar, but the strains 3T-1 and MJ-102 contained more repetitive sequences. Similarly, there are similarities between their virulence factors, but there are also some differences. In addition, the results of collinearity analysis showed that 3T-1 and MJ-102 had lower similarity and longer evolutionary distance with Rhs1AP, but the genetic relationship between 3T-1 and MJ-102 was closer. This study can lay a foundation for studying the molecular pathogenesis and virulence factors of R. solani AG-3, and revealing its genomic composition will also help to develop more effective disease control strategies.

Effects of Chemical and Biological Fungicide Applications on Sexual Sporulation of Rhizoctonia solani AG-3 TB on Tobacco.

Rhizoctonia solani AG-3 TB primarily causes tobacco target spot disease by producing a large number of sexual spores. However, inducing sexual spore formation under in vitro conditions has been challenging, impeding further research on its control. In this study, field experiments were conducted to assess the effects of different concentrations of chemical and biological fungicides on the production of sexual spores of R. solani AG-3 TB on tobacco plants. The results demonstrated that four chemical fungicides (propiconazole-morpholine guanidine, bordeaux mixture, thiophanate-methyl, and mancozeb) significantly induced sexual spore formation. Among them, increasing the concentrations of the first three fungicides resulted in an increase in the number of sexual spores, while increasing the concentration of mancozeb led to a decrease in spore count. The pathogenic fungus produced more sexual spores during the night than during the day. Temperature, humidity, and light conditions influenced spore production. Additionally, the infection rate of sexual spores was directly proportional to their concentration and inoculation time, but their survival time did not exceed 6 h in vitro. Importantly, Streptomyces rectiolaceus A8 significantly suppressed sexual spore formation, achieving an 83.63% control efficacy in the field and producing antimicrobial substances against R. solani AG-3 TB. In conclusion, appropriate concentrations of chemical fungicides can induce sexual spore formation, while A8 can inhibit their production, showing potential value for controlling tobacco target spot disease.

Argonaute and Dicer are essential for communication between Trichoderma atroviride and fungal hosts during mycoparasitism.

Trichoderma species are known for their mycoparasitic activity against phytopathogenic fungi that cause significant economic losses in agriculture. During mycoparasitism, Trichoderma spp. recognize molecules produced by the host fungus and release secondary metabolites and hydrolytic enzymes to kill and degrade the host's cell wall. Here, we explored the participation of the Trichoderma atroviride RNAi machinery in the interaction with six phytopathogenic fungi of economic importance. We determined that both Argonaute-3 and Dicer-2 play an essential role during mycoparasitism. Using an RNA-Seq approach, we identified that perception, detox, and cell wall degradation depend on the T. atroviride-RNAi when interacting with Alternaria alternata, Rhizoctonia solani AG2, and R. solani AG5. Furthermore, we constructed a gene co-expression network that provides evidence of two gene modules regulated by RNAi, which play crucial roles in essential processes during mycoparasitism. In addition, based on small RNA-seq, we conclude that siRNAs regulate amino acid and carbon metabolism and communication during the Trichoderma-host interaction. Interestingly, our data suggest that siRNAs might regulate allorecognition (het) and transport genes in a cross-species manner. Thus, these results reveal a fine-tuned regulation in T. atroviride dependent on siRNAs that is essential during the biocontrol of phytopathogenic fungi, showing a greater complexity of this process than previously established.IMPORTANCEThere is an increasing need for plant disease control without chemical pesticides to avoid environmental pollution and resistance, and the health risks associated with the application of pesticides are increasing. Employing Trichoderma species in agriculture to control fungal diseases is an alternative plant protection strategy that overcomes these issues without utilizing chemical fungicides. Therefore, understanding the biocontrol mechanisms used by Trichoderma species to antagonize other fungi is critical. Although there has been extensive research about the mechanisms involved in the mycoparasitic capability of Trichoderma species, there are still unsolved questions related to how Trichoderma regulates recognition, attack, and defense mechanisms during interaction with a fungal host. In this work, we report that the Argonaute and Dicer components of the RNAi machinery and the small RNAs they process are essential for gene regulation during mycoparasitism by Trichoderma atroviride.

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 Rhizoctonia solani AG-4 HG-I Causing Root and Basal Stem Rot on Roselle (Hibiscus sabdariffa) in Mexico.

Roselle (Hibiscus sabdariffa L.) is a crop of economic importance, refreshing drinks are prepared from its calyces, it is also attributed to antioxidant, antibacterial, and antihypertensive properties (Da-Costa-Rocha et al. 2014). In November 2022, in municipality of Iguala (18.355592N, 99.548546W, 749 m above sea level), Guerrero, México, roselle plants of approximately 1.5 months of age with basal rot were detected under greenhouse conditions. The symptoms consisted of wilting, yellowing, and root and stem rot with constriction in the base of the stem. The symptoms were detected in approximately 15% of plants at the operation. From symptomatic tissue, cuts were made into approximately 0.5 cm pieces, sterilized with 2% NaClO, washed with sterile distilled water, transferred to PDA medium amended with 50 mg/liter of Chloramphenicol, and incubated in the dark for four days at 28 °C. Rhizoctonia-like colonies were consistently obtained, and nine isolates were selected and purified by the hyphal-tip method. After four days, isolates developed a mycelium was light-white that became brown with age. Right-angled hyphal branching was also observed, in addition to a slight constriction at the base of the branches. In some older cultures, numerous dark brown sclerotia were observed. They were multinucleate cell with three to eight nuclei and measured from 1 to 2 mm in diameter. Together these characteristics were consistent with the description of Rhizoctonia solani Kühn (Parmeter 1970). The anastomosis group (AG) was confirmed by amplifying the ITS region with the primers ITS1 and ITS4 (White et al. 1990) of the RIJAM3 and RIJAM5 strains. The sequences were deposited in GenBank (Nos. OR364496 and OR364497 for RIJAM3 and RIJAM5, respectively). BLAST analysis, both isolates indicated 99.7 identity to R. solani AG-4 HG-I (GenBank: KM013470) strain ICMP 20043 (Ireland et al. 2015). The phylogenetic analysis of AGs sequences allowed assignment of isolates RIJAM3 and RIJAM5 to the AG-4 HG-1 clade. A pathogenicity test was performed on 20 one-month-old roselle plants. Mycelium of RIJAM3 isolate was inserted into the base of the stem with a sterile toothpick. As a control, a sterile toothpick with no mycelium was inserted in ten healthy plants. Additionally, 50 eight-day-old seedlings were inoculated by placing a 5-mm diameter agar plug colonized with mycelium of RIJAM3 at the base of the stem 10 mm below the soil surface. As control treatments, uncolonized PDA plugs were deposited at the base of 25 seedlings. The inoculated plants were incubated in a greenhouse with an average temperature and relative humidity of 28°C and 85%, respectively. Following inoculation, symptoms similar to those observed in the original outbreak were observed in plants after six days and only after four days in seedlings. In both experiments, the control plants and seedlings remained asymptomatic. R. solani was re-isolated from plants and seedlings, complying with Koch's postulates. The pathogenicity testing was repeated twice, with concordant results. In Nigeria and Malaysia R. solani was reported to seedling death to cause seedling dieback in roselle (Adeniji 1970; Eslaminejad and Zakaria 2011). In México R. solani AG-4 has been previously reported in crops of potato, chili and tomato (Montero-Tavera et al. 2013; Ortega-Acosta et al. 2022; Virgen-Calleros et al. 2000). To the best of our knowledge, this is the first report of R. solani AG-4 HG-I as a causing of root and basal stem rot on roselle in Mexico. This research provides information essential for informing the management of this disease, and may help design measures to prevent the spread of the pathogen to other regions.

Isolation, identification, and pathogenicity of Rhizoctonia spp. recovered from soil samples of the greenhouse tomato growing area of the West Mediterranean region, Türkiye

AbstractThis study's objective was to identify the anastomosis groups and pathogenicity of Rhizoctonia species from soil samples in tomato production areas of Antalya, Burdur, and Isparta provinces in the West Mediterranean Region of Türkiye during the 2022 season. Soil samples were gathered from tomato plant rhizospheres in 223 different greenhouses, and isolations were done from the soil using the baiting technique. According to the results of morphological characterization and sequence analysis of the internal transcribed spacers of the ribosomal DNA, 47 Rhizoctonia isolates were defined as binucleate and 21 isolates as multinucleate. The collection of binucleate Rhizoctonia (Ceratobasidium sp.) isolates included representatives of Anastomosis Group‐F (61.76%), AG‐A (1.47%), and an unidentified binucleate Rhizoctonia (Ceratobasidium sp.) (5.88%). Multinucleate R. solani included AG 4 HG‐I (26.47%) and AG 4 HG‐II (4.41%). Pathogenicity tests showed statistically significant differences in virulence between binucleate Rhizoctonia (Ceratobasidium sp.) isolates and multinucleate Rhizoctonia isolates. All R. solani AG 4 HG‐II isolates and most of the AG 4 HG‐I isolates were pathogenic, causing severe necrosis symptoms and damping off in tomato seedlings, but binucleate AG‐A and AG‐F isolates were nonpathogenic. This study is the first detailed report about Rhizoctonia species isolated from the soil of tomato plants grown in greenhouse production areas in Türkiye.

Binucleate Rhizoctonia induced tomato resistance against Rhizoctonia solani via affecting antioxidants and cell wall reinforcement

Isolates of Rhizoctonia solani (AG-3 PT, AG-4 HG-I, AG-4 HG-II) and one binucleate Rhizoctonia sp. (BNR) belonging to AG-Bb were investigated for pathogenicity on tomato cultivar Mobil. The BNR isolate revealed the lowest virulence and it was used as biocontrol agent against R. solani AG-4 HG-II, which showed the highest virulence on tomato. Inoculation of tomato plants with the hypovirulent BNR isolate reduced the disease symptoms of R. solani and induced resistance. Resistance induction was observed not only on the plants simultaneously inoculated with BNR and R. solani, but also when the plants were inoculated by the BNR and R. solani with time intervals. The peroxidase (POX), superoxide dismutase (SOD) and catalase (CAT) activities and expression levels of the corresponding genes in tomato plants increased after R. solani or BNR inoculation. The highest level of antioxidant activities and expression of their genes, lignin and callose formation were observed in the plants inoculated with the BNR and R. solani, simultaneously. The BNR inoculation reduced H2O2 accumulation. The highest level of priming was observed for the POX among other antioxidants tested via application of the BNR. Treatment with potassium cyanide (as a POX inhibitor) reduced basal resistance and BNR-induced resistance (BNR-IR) via reduction of lignification and callose deposition in tomato plants. These findings demonstrated the role of antioxidant enzymes, mainly the POX, in both basal resistance and BNR-IR. Therefore, redox state and antioxidants are involved in cell wall strengthening via lignin and callose formation, as important defense components which decrease the pathogen progress in plant tissues.

Супресивен ефект на почвата към ризоктонийно кореново гниене, индуциран чрез имитирана монокултура на пшеница

Disease decline and induction of soil suppressiveness by continuous (5-6 years) monoculture with susceptible plant-host have been reported for various crop-pathogen systems. The present study achieves the goal of inducing suppressive effect in soil towards Rhizoctonia root rot (Rhizoctonia solani AG 4) through monoculture of winter wheat (Triticum aestivum L.). Twenty four high-quality wheat (c. Exotic) seeds were planted in plastic containers, each containing 1.1 L Alluvial-Meadow (sandy-clay) soil (Eutric Fluvisols, according to FAO classification). According to the treatment applied, the resulting plants were grown for 0-4 successive short cropping cycles, each lasting three months. Disease suppressiveness was measured in a bioassay in which the experimental units were rectangular, plastic containers (26 x 6 x 8 cm), with drainage holes in the bottom and detachable trays allowing for manual irrigation from the bottom of the container, 7 cm height of the column of tested soil in each container and a two-row crop of wheat (c. Exotic, 2 rows x 12 plants, 2 x 3 cm plant spacing). The spread of the disease was measured from a focus (point) source of inoculum of the pathogen – 20 inoculum grains from a pure culture developed on autoclaved (30 min at 121 °C) barley, introduced into the soil at the beginning of both rows after emergence of the plants. Immediately after termination of the soil suppressiveness test, the abundance of the major groups of soil microorganisms were determined in rhizosphere soil in each treatment by using the method of decimal dilutions and subsequent culturing on agar media. Separately, bacteria of the genus Pseudomonas were isolated from the rhizosphere soil on King’s medium. The antagonistic activity of 10 Pseudomonas spp. isolates from each treatment was tested against R. solani AG 4 in vitro by the double culture method. The monoculture of wheat imitated under controlled conditions resulted in a significant (P<0.05) reduction of the spread of Rhizoctonia root rot from the source of infection. Furthermore, statistically significant differences (P<0.05) were also found between the individual treatments with pre-cultivation of wheat: with the increase in the number of cultivation cycles from one to four, there was a significant reduction in disease spread – by 19.5%, 31.0%, 40.5% and 54.8%, respectively, as compared to the untreated control. Simultaneously, no substantial alterations were observed in the population densities of the monitored groups of microorganisms in the rhizosphere of the experimental plants, including ammonifying and spore-forming bacteria, microscopic fungi, Actinomycetes and bacteria, utilizing mineral N. With the increase in soil suppressiveness from the first to the fifth growing cycle of the crop, a significant increase in the average antagonistic activity of the isolated Pseudomonas spp. against R. solani AG 4 was observed. The thesis is argued that shattering rachis of the ripe ear in wild forms conditioned a continuous “natural” monoculture in which bread wheat and its predecessors grew and evolve during the prehistoric era. We speculate that this condition, along with the lack of genes for resistance to necrotrophic pathogens, have given rise to evolutionary stable defence mechanism and the latter based on mutualistic relationships between the host-plant and bacteria that naturally inhabit rhizosphere and exhibiting high antagonistic (antibiotic) activity against soil-borne plant pathogens. A manifestation of this mechanism is the increasing suppressive response of the soil and the disease decline during or after continuous wheat monoculture. The results obtained in this study are discussed in the light of the microbial eco-evolutionary dynamics in the plant rhizosphere. The progressively increasing pathogen-antagonistic activity of Pseudomonas spp. isolates is discussed as an example of “rapid microbial evolution”, accomplished by possible horizontal gene transfer within one or more successive cultivation cycles of the wheat crop.

Identification and nucleotide sequencing of a novel partitivirus derived from Rhizoctonia solani AG-4 HG III isolate A14.

Rhizoctonia solani is a widely disseminated phytopathogen that is found in the soil and is capable of harming many important species of crops. Here, analysis of the R. solani AG-4 HG III strain A14 led to the identification of a novel mycovirus assigned the tentative name "Rhizoctonia solani partitivirus A14" (RsPV-A14), which was subjected to sequencing and associated analyses. This approach revealed that RsPV-A14 harbored two dsRNA segments, 2022 bp (dsRNA1) and 1905 bp (dsRNA2) in length. dsRNA1 was found to contain a single open reading frame (ORF1) that codes for a 622-amino-acid protein with conserved RNA-dependent RNA polymerase (RdRp) motifs, and dsRNA2 was found to contain an ORF (ORF2) that is predicted to code for a 558-amino-acid capsid protein (CP). BLASTp analysis using the putative RdRp of RsPV-A14 showed sequence similarity to partitiviruses, including Rosellinia necatrix partitivirus 7 (50.53% identity), an unclassified partitivirus. Phylogenetic analysis based on RdRp protein sequences suggested that RsPV-A14 is a novel member of the family Partitiviridae.

Identification and physiological activity of (methoxymethyl)triphenylphosphonium chloride as a new phytotoxin isolated from Rhizoctonia solani AG-3 TB.

Rhizoctonia solani as a cosmopolitan fungus is the causative agent of many crop diseases and leads to significant economic losses in crop production. To explore the toxin structure and physiological activity of R. solani AG-3 TB, high-performance liquid chromatography (HPLC), infrared absorption spectrum (IR), and nuclear magnetic resonance spectrum (NMR) were required. Here, the compound (methoxymethyl)triphenylphosphonium chloride (MMC) with the molecular formula C20H20ClOP was purified and identified from R. solani AG-3 TB. The pure compound MMC treated at 20 μg/mL, 50 μg/mL, and 100 μg/mL can cause obvious necrosis on leaves, increase active oxygen species (AOS), decrease chlorophyll content, and damage cellular structure. The results enrich the understanding of toxin compounds for R. solani and provide valuable insights into the toxicology of R. solani AG-3 TB.

The role of oxidative burst, antioxidant genes and enzymes in association with callose in tomato reaction to various taxonomic groups of Rhizoctonia spp.

Various fungi are significant threats to vegetable yield via causing destructive diseases. Rhizoctonia solani is a soilborne fungal pathogen affecting tomato with considerable damage in crop production, worldwide. Understanding plant defense mechanisms against different Rhizoctonia spp. isolates can be useful in designing novel and effective disease control approaches. Therefore, we performed cellular, molecular and biochemical assays to investigate the interaction of various taxonomic groups of multinucleate R. solani and a binucleate isolate of Rhizoctonia sp. (BNR) with tomato as a susceptible host. Histochemical and enzymatic experiments showed that the highest levels of O2−, H2O2, callose accumulation and superoxide dismutase activity were observed for the isolate of R. solani AG 3, which caused lower disease severity compared to the highly pathogenic R. solani isolates belonging to AG 4 HG-I and AG 4 HG-II. Gene expression investigations via quantitative reverse transcription-polymerase chain reaction revealed the highest levels of the SOD gene upregulation in the plants with R. solani AG 3 and the BNR inoculations, which showed the lowest levels of pathogenicity on tomato plants. Whereas, the highest upregulation of the POX expression was detected in tomato plant infected with the highly pathogenic R. solani isolates belonging to AG 4 HG-I and AG 4 HG-II at most of the time points investigated. Xanthine/xanthine oxidase treatment, which induced O2− production, increased the disease symptoms. But, glucose/glucose oxidase, which increased H2O2, decreased the disease symptoms. Ascorbate, as an inhibitor of H2O2 accumulation, reduced callose deposition and increased the disease severity. In overall, upregulation of the SOD gene expression and activation of the corresponding enzyme, which are linked to H2O2 and callose accumulation, might be related to lower level of the disease progress on the plants inoculated with R. solani AG 3 and the BNR isolates. So, priming these defense mechanisms via different environmentally safe treatments could be helpful in disease management, which needs further investigations in future.

Mitochondrial-targeting effector RsIA_CtaG/Cox11 in Rhizoctonia solani AG-1 IA has two functions: plant immunity suppression and cell death induction mediated by a rice cytochrome c oxidase subunit.

Rhizoctonia solani AG-1 IA causes a necrotrophic rice disease and is a serious threat to rice production. To date, only a few effectors have been characterized in AG-1 IA. We previously identified RsIA_CtaG/Cox11 and showed that infiltration of the recombinant protein into rice leaves caused disease-like symptoms. In the present study, we further characterized the functionality of RsIA_CtaG/Cox11. RsIA_CtaG/Cox11 is an alternative transcript of cytochrome c oxidase copper chaperone Cox11 that starts from the second AUG codon, but contains a functional secretion signal peptide. RNA interference with RsIA_CtaG/Cox11 reduced the pathogenicity of AG-1 IA towards rice and Nicotiana benthamiana without affecting its fitness or mycelial morphology. Transient expression of the RsIA_CtaG/Cox11-GFP fusion protein demonstrated the localization of RsIA_CtaG/Cox11 to mitochondria. Agro-infiltration of RsIA_CtaG/Cox11 into N. benthamiana leaves inhibited cell death by BAX and INF1. In contrast to rice, agro-infiltration of RsIA_CtaG/Cox11 did not induce cell death in N. benthamiana. However, cell death was observed when it was coinfiltrated with Os_CoxVIIa, which encodes a subunit of cytochrome c oxidase. Os_CoxVIIa appeared to interact with RsIA_CtaG/Cox11. The cell death triggered by coexpression of RsIA_CtaG/Cox11 and Os_CoxVIIa is independent of the leucine-rich repeat receptor kinases BAK1/SOBIR1 and enhanced the susceptibility of N. benthamiana to AG-1 IA. Two of the three evolutionarily conserved cysteine residues at positions 25 and 126 of RsIA_CtaG/Cox11 were essential for its immunosuppressive activity, but not for cell death induction. This report suggests that RsIA_CtaG/Cox11 appears to have a dual role in immunosuppression and cell death induction during pathogenesis.

Characterization of heteropolysaccharides from Rhizoctonia solani AG1 IA cell wall and comparison of their effect on inducing plant defense

Rhizoctonia solani (R. solani) is an important pathogenic fungus that causes symptoms of sheath blight, and the polysaccharide-rich cell wall plays a major role in plant-pathogen interactions. However, the composition and structure of its cell wall polysaccharides are insufficiently understood, and its specific function in plant-pathogen interactions is unknown, which makes effective control of sheath blight difficult at present. Herein, five cell wall polysaccharides (WF-1, WF-2, CAF-1, HAF-1 and HAF 2-1) were sequentially extracted by boiling water, cold and hot alkali from R. solani AG1 IA. They were heteropolysaccharides containing mainly glucose, mannose and galactose and less fucose, with molecular weights above 1100 kDa. These five polysaccharides mainly composed of →4)-Glcp-(1→, →6)-Glcp-(1→, →4,6)-Glcp-(1→, →3,4)-Glcp-(1→, and Manp-(1→. Several polysaccharides, except WF-1, showed different induced resistance degrees on rice plant, with HAF 2-1 having the most significant effect. Further analysis using NMR confirmed that the backbone of HAF 2-1 mainly consisted of →4)-α-D-Glcp-(1→ and →6)-α-D-Glcp-(1→ with branches of →4,6)-D-Glcp-(1→. HAF 2-1 enhance the resistance of rice against R. solani through salicylic acid (SA)-mediated immune signaling pathway. This work improves our knowledge of the cell wall polysaccharides in plant pathogens and facilitates the study of pathogenic mechanisms and effective disease control.

Horizontal and Vertical Transmission of a Mycovirus Closely Related to the Partitivirus RhsV717 That Confers Hypovirulence in Rhizoctonia solani.

Rhizoctonia solani virus717 (RhsV717) was isolated from the Rhizoctonia solani (R. solani) AG-2 strain Rhs717. This study isolated a virus designated as Rhizoctonia solani partitivirus BS-5 (RsPV-BS5) from the R. solani AG-3 strain BS-5, the causal agent of tobacco target spot disease. The virus was identified as a strain of RhsV717. Transmission electron microscopy (TEM) images showed that RsPV-BS5 had virus particles with a diameter of approximately 40 nm. Importantly, it can be horizontally transmitted through hyphal anastomosis and vertically transmitted via sexual basidiospores. Furthermore, this study demonstrated that RsPV-BS5 infection significantly impedes mycelial growth and induces hypovirulence in tobacco leaves. Thus, RsPV-BS5 presents a promising avenue for biocontrolling tobacco target spot disease. Transcriptome analysis unveiled differential expression of four genes related to cell wall-degrading enzymes between two isogenic strains, 06-2-15V and 06-2-15. These findings shed light on the molecular mechanism through which RsPV-BS5 reduces host pathogenicity.