First Report of Binucleate Rhizoctonia AG-G Causing Root Rot of Japanese Bay Tree (Machilus thunbergii) in Korea.

Machilus thunbergii Sieb. & Zucc., commonly known as Japanese bay tree, is a large evergreen tree belonging to the Lauraceae family and is widely distributed in Asia, including Korea in subtropical and tropical forest areas (Wu et al., 2006). In April 2021, a root rot disease of 2-year-old Japanese bay trees was observed in a nursery on Wando Island in Korea. Tree roots exhibited brown to black discoloration, root rot, and deterioration, and leaves were severely wilted followed by plant death, with a disease incidence of approximately 30%. Symptomatic roots were surface sterilized with 1% NaOCl for 5 min and washed three times with distilled water. The root tissues were dried and plated on potato dextrose agar (PDA) and vegetable juice agar (V8) media. After 3-4 days of incubation at 25 ˚C, brown Rhizoctonia fungal-like colonies grew on both culture media. Hyphae of two representative isolates (CMML21-35 and CMML21-36) exhibited typical characteristics of Rhizoctonia, including a constriction of branching hyphae (Alvarez et al., 2013). In addition, two nuclei in each mycelial cell were observed after staining of mycelia with 0.1% Safranin O. The two isolates were identified as binucleate Rhizoctonia based on the microscopic observation. To confirm identification of the isolates, the internal transcribed spacer (ITS) and large subunit (LSU) regions were sequenced using two primer sets, ITS1/ITS4 and LROR/LR5 (White et al., 1990; Vilgalys and Hester 1990). BLASTn search analysis revealed that the ITS sequence of isolates had 99.66% (582 base pair matched of 584) sequence similarity with the sequences of binucleate Rhizoctonia (accession numbers JF519837 and AY927327, respectively) and the LSU sequence matched well with the sequence of Rhizoctonia sp. AG-G (accession number MN977413; similarity 99.56% and 910 base pair matched of 914). The sequences were deposited in GenBank under accession numbers OM049427 and OM049428 for ITS, OM679289 and OM679290 for LSU. Phylogenetic analysis of ITS and LSU regions revealed that the isolates grouped with binucleate Rhizoctonia anastomosis group AG-G (Teleomorph: Ceratobasidium sp.) with a high bootstrap value. Accordingly, the morphological and molecular characteristics confirmed the causal pathogen as binucleate Rhizoctonia AG-G (Jiang et al., 2016; Gonzalez et al. 2016). To test pathogenicity, a 2-year-old Japanese bay tree was inoculated by creating a hole in the soil near the root rhizosphere and placing 1.5g of ground mycelia obtained from a 5 day-old broth culture at two time points one week apart (Bartz et al., 2010). The control pot was inoculated with sterilized ddH2O. Inoculated and control plant pots were incubated in plastic boxes with 100% relative humidity at 25 ℃ for five days. After that, the pots were placed in the greenhouse at 23-25 ℃. One month post inoculation, initial disease symptoms were observed, and after two months, severe foliar wilting and eventual plant death occurred. The non-inoculated control remained healthy. The pathogen was re-isolated from infected roots, fulfilling Koch's postulates. The experiment was conducted three times with three replications. This is the first report of root rot of Japanese bay tree caused by binucleate Rhizoctonia AG-G in Korea and in the world. Previously, a pathogenic binucleate Rhizoctonia AG-G was isolated from colonized apple tree roots in orchards in Italy (Kelderer et al., 2012). The present study implies that this pathogen potentially causes a negative impact on the nursery and forest industries, thus further research on the screening for pathogenicity in other tropical and subtropical trees and also apple, which is an important crop in Korea, is needed.

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.

Diversity of Mycoviruses Present in Strains of Binucleate Rhizoctonia and Multinucleate Rhizoctonia, Causal Agents for Potato Stem Canker or Black Scurf.

In this study, the diversity of putative mycoviruses present in 66 strains of binucleate Rhizoctonia (BNR, including anastomosis group (AG)-A, AG-Fa, AG-K, and AG-W) and 192 strains of multinucleate Rhizoctonia (MNR, including AG-1-IA, AG-2-1, AG-3 PT, AG-4HGI, AG-4HGII, AG-4HGIII, and AG-5), which are the causal agents of potato stem canker or black scurf, was studied using metatranscriptome sequencing. The number of contigs related to mycoviruses identified from BNR and MNR was 173 and 485, respectively. On average, each strain of BNR accommodated 2.62 putative mycoviruses, while each strain of MNR accommodated 2.53 putative mycoviruses. Putative mycoviruses detected in both BNR and MNR contained positive single-stranded RNA (+ssRNA), double-stranded RNA (dsRNA), and negative single-stranded RNA (-ssRNA) genomes, with +ssRNA genome being the prevalent nucleic acid type (82.08% in BNR and 75.46% in MNR). Except for 3 unclassified, 170 putative mycoviruses found in BNR belonged to 13 families; excluding 33 unclassified, 452 putative mycoviruses found in MNR belonged to 19 families. Through genome organization, multiple alignments, and phylogenetic analyses, 4 new parititviruses, 39 novel mitoviruses, and 4 new hypoviruses with nearly whole genome were detected in the 258 strains of BNR and MNR.

PCR-DGGE Analysis Proves the Suppression of Rhizoctonia and Sclerotium Root Rot Due to Successive Inoculations.

The soil-borne pathogens Rhizoctonia solani and Sclerotium rolfsii have emerged as major pathogens of radish (Raphanus sativus) worldwide. The induction of soil suppressive of radish root rot disease was evaluated in soil repeatedly inoculated with R. solani, nonpathogenic binucleate Rhizoctonia sp. AG-A W1 (BNR) and S. rolfsii. The repeated inoculations of soil with R. solani and BNR significantly suppressed the disease severity of R. solani and S. rolfsii compared to the control. In contrast, the repeated inoculation of soil with S. rolfsii significantly suppressed only the pathogen, S. rolfsii. The community structure was examined using PCR-DGGE (polymerase chain reaction denaturing gradient gel electrophoresis) method. The bands of Trichoderma sp. were observed in the first, second and third inoculations of the soil with BNR. Similarly, bands of Trichoderma sp. were observed in the second and third inoculations of the soil with S. rolfsii and R. solani. Compared to the control, disease severity was significantly reduced in the soil repeatedly inoculated with S. rolfsii and R. solani . In conclusion, Trichoderma species were accumulated in specific patterns depending on the applied fungal inoculum in the suppressive soil.

Induction of Systemic Resistance in Cucumber by Hypovirulent Binucleate Rhizoctonia against Anthracnose Caused by Colletotrichum orbiculare.

Treatment with hypovirulent binucleate Rhizoctonia (HBNR) isolates induced systemic resistance against anthracnose infected by Colletotrichum orbiculare in cucumber, as there were no direct interaction between HBNR and C. orbiculare. This is because of the different distances between HBNR and C. orbiculare, where the root was treated with HBNR isolate and C. orbiculare was challenged and inoculated in leaves or first true leaves were treated with HBNR isolate and C. orbiculare was challenged and inoculated in second true leaves. The use of barley grain inocula and culture filtrates of HBNR significantly reduced the lesion diameter compared to the control (p = 0.05). The total lesion diameter reduction by applying barley grain inoculum of HBNR L2, W1, W7, and Rhv7 was 28%, 44%, 39%, and 40%, respectively. Similar results was also observed in treatment using culture filtrate, and the reduction of total lesion diameter by culture filtrate of HBNR L2, W1, W7, and Rhv7 was 45%, 46%, 42%, and 48%, respectively. When cucumber root was treated with culture filtrates of HBNR, the lignin was enhanced at the pathogen penetration, which is spread along the epidermis tissue of cucumber hypocotyls. Peroxidase activity in hypocotyls in the treated cucumber plant with culture filtrates of HBNR significantly increased before and after inoculation of pathogens as compared to the control. Significant enhancement was also observed in the fast-moving anodic peroxidase isozymes in the treated plants with culture filtrates of HBNR. The results showed the elicitor(s) contained in culture filtrates in HBNR. The lignin deposition as well as the peroxidase activity is an important step to prevent systemically immunised plants from pathogen infection.

Seasonal dynamics and fungicide sensitivity of organisms causing brown patch of tall fescue in North Carolina.

Brown patch, caused by multiple species of Rhizoctonia and Rhizoctonia-like fungi, is the most severe summer disease of tall fescue in home lawns across the southeastern United States. Home lawns were surveyed in central North Carolina from 2013 to 2015 to determine the organisms present during typical epidemics of brown patch in tall fescue. Isolates of Rhizoctonia and Rhizoctonia-like fungi were obtained by sampling 147 locations in July 2013 and May and July 2014. In addition, 11 sites were sampled once a week for 12 consecutive weeks from late May to the end of July 2015. All isolates were identified to species and anastomosis group with nuc rDNA internal transcribed spacer (ITS) sequence analysis. Isolations from brown patch lesions in May 2014 predominately yielded Ceratobasidium cereale (77% of the organisms recovered), whereas the organisms recovered in July 2013 and 2014 were R. solani AG 2-2-IIIB (44%), R. solani AG 1-IB (37%), and R. zeae (14%). In 2015, Ceratobasidium cereale was isolated from all 11 locations in May but was replaced by Rhizoctonia species in June and July. Sensitivity of the May 2014 isolates to multiple concentrations of the fungicides azoxystrobin, flutolanil, fluxapyroxad, and propiconazole was compared with sensitivity of isolates collected in 2003, to determine whether multiple years of exposure to fungicides applied for brown patch control had altered fungicide sensitivity. Historical isolates of R. solani, which had never been exposed to fungicide applications for brown patch control, were also included for comparison. Mean EC50 values (concentration of fungicide needed to inhibit mycelial growth by 50%) varied across fungicides and species, but no resistance was observed, and there was no apparent shift in sensitivity over the years. An additional 94 isolates from 2015 were screened against azoxystrobin, flutolanil, fluxapyroxad, and propiconazole, and fungicide insensitivity was not observed.