Anastomosis groups and pathogenicity of binucleate Rhizoctonia isolates associated with stem canker of potato in China

Potato stem canker caused by Rhizoctonia solani commonly occurs in potato-growing regions around the world, but little is known about whether binucleate Rhizoctonia (BNR) can incite this disease on potato plants in China. In the present study, a total of 69 BNR isolates were recovered from cankered subterraneous stems of potato collected from 17 provinces, autonomous regions, and municipalities of China. Based on the morphological characteristics and sequence analysis of the internal transcribed spacers of the ribosomal DNA, 48 isolates were classified as anastomosis group (AG)-A with a ratio of 69.56 %, 15 isolates (21.74 %) were AG-K, four isolates (5.80 %) were AG-F, one isolate (1.45 %) was AG-I and one isolate (1.45 %) was AG-U. Pathogenicity tests under glasshouse conditions revealed that all BNR isolates, except for the AG-I isolate, could induce symptoms of stem canker on potato plants with disease incidence ranging from 5.26 to 55.56 % and disease index ranging from 1.32 to 13.89, respectively. To our knowledge, this is the first detailed report about BNR AGs causing stem canker on potato plants in China.     

Clostridium bifermentans and C. subterminale are associated with kiwifruit vine decline,known as moria,in Italy

Since 2012, a new pathogenic syndrome has frequently been observed in many areas of kiwifruit cultivation in Italy. The main symptoms include an initial withering of the leaves followed by a total and sudden collapse of plants, mainly occurring during summer. The withered leaves fall and the main and secondary feeder roots appear rotten, sometimes showing a reddish-brown discoloration. The disease, that affects both the green and yellow-fleshed cultivars, has been called kiwifruit vine decline and is locally known as moria. The syndrome has been found consistently associated with soil waterlogging, which frequently occurs either after the traditional agronomical practice of irrigating orchards through surface irrigation or after very heavy rainfall. So far, the role played by bacteria in this syndrome has not been investigated. In the present study, Clostridium spp. were isolated from both rotten roots and soils obtained from Italian kiwifruit orchards affected by the syndrome, indicating for the first time that anaerobic bacteria are able to cause damage to woody crops. C. bifermentans and C. subterminale incited symptoms in kiwifruit in both in vivo and in vitro pathogenicity tests. Soil waterlogging seems to potentially favour colonization of kiwifruit roots by anaerobic bacteria, probably because saturation of the soil can facilitate proliferation and persistence of these bacteria during long periods of the vegetative growth of the crop. The occurrence of anaerobic bacteria does not exclude the possibility that other microorganisms can play additional/synergic role(s) in causing the kiwifruit vine decline.     

Characterization and pathogenicity of Rhizoctonia isolates associated with cauliflower in Belgium

Sixty-two isolates of Rhizoctonia spp. were collected from Belgian cauliflower fields during 2005 and 2006. The majority of the isolates (60 out of 62) had multinucleate cells and were identified as Rhizoctonia solani . Characterization of anastomosis groups (AGs) was performed using pectic zymograms, PCR-RFLP and sequencing of the rDNA-ITS region. The most prevalent AG was AG 2-1 (55% of isolates), followed by AG 2-1 subset Nt (11%), AG 1-1C (8%), AG 5 (8%), AG 4 HGII (6%), AG 3 (5%) and AG 1-1B (3%). Pathogenic potential towards different vegetable crops and towards maize was determined. Damage to cauliflower and endive was caused by different AGs, with the isolates aggressive towards cauliflower belonging to AG 2-1, AG 2-1 subset Nt, AG 4 HGII, AG 1-1C, AG 1-1B and AG 2-2, and those aggressive towards endive belonging to AG 1-1B, AG 1-1C, AG 2-1 subset Nt, AG 2-2, AG 4 HGII and AG 5. The most aggressive isolates towards bean belonged to AG 2-1 subset Nt and AG 2-2, for lettuce to AG 1-1B and AG 2-1, on carrot to AG 4 HGII and towards maize to AG 2-2. Within the isolates of AG 2-1, variability was observed in PCR-RFLP pattern and in aggressiveness towards several crops, indicating this subgroup to be heterogeneous. This is the first study concerning the occurrence of R. solani AGs causing wirestem in Belgian cauliflower fields and the first report of aggressive isolates of AG 1-1C, AG 2-1 subset Nt and AG 4 HGII associated with cauliflower.     

rDNA-based characterization of a new binucleate Rhizoctonia spp. causing root rot on kale in Brazil

In this paper we present the first report of the occurrence of a binucleate Rhizoctonia spp. causing hypocotyl and root rot in kale in Brazil. Rhizoctonia spp. were isolated from kale (Brassica oleracea var. acephala) with symptoms of hypocotyl and root rot. The isolates, characterized as binucleate Rhizoctonia spp., did not show an anastomosis reaction with any of the binucleate Rhizoctonia spp. testers used. The pathogenicity of the isolates was tested under greenhouse conditions; all isolates were pathogenic and showed different symptom severities on kale. The ITS-5.8S rDNA sequences of kale isolates and 50 testers (25 binucleate Rhizoctonia spp. and 25 Rhizoctonia solani) were compared in order to characterize the genetic identity of Rhizoctonia spp. infecting kale. The kale isolates showed genetic identities ranging from 99.3 to 99.8% and were phylogenetically closely related to CAG 7 (AF354084), with identities of 98.5 and 98.7%. It is suggested that the binucleate Rhizoctonia spp. causing hypocotyl and root rot on kale Brazil comprises a new AG not yet described.     

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

In 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.     

Evaluation of Brassica species for resistance to Rhizoctonia solani and binucleate Rhizoctonia (Ceratobasidum spp.) under controlled environment conditions

Isolates of R. solani AG 2–1, AG 8, AG 10 and binucleate Rhizoctonia (Ceratobasidium spp.) were tested for virulence on Brassica crops in growth chamber experiments. Isolate virulence and genotype resistance were determined based on percent of seedling survival, shoot length reduction, and shoot fresh weight. Isolates had significant effects on all tested measurements, compared to the non-inoculated controls. Rhizoctonia solani AG 2–1 appears to be the most aggressive pathogen on all tested genotypes followed by R. solani AG 8, binucleate Rhizoctonia and R. solani AG 10, respectively. Genotype by isolate interaction effects were found to be significant for percent of seedling survival and shoot length reduction. None of the tested genotypes exhibited any level of resistance to R. solani AG 2–1, but three promising genotypes with moderate levels of resistance to R. solani AG 10, R. solani AG 8 and binucleate Rhizoctonia were identified. Moderate heritability (0.57) was observed for the percent of seedling survival in the resistant genotype KS4022.     

Molecular identification of Fusarium spp. associated with bakanae disease of rice in Italy and assessment of their pathogenicity

Between 2006 and 2008, 146 isolates of Fusarium spp. were obtained from bakanae‐diseased rice plants and seeds from the major rice‐growing regions of Italy. These isolates were identified based on translation elongation factor (EF‐1α) sequence and pathogenicity tests were used to assess their aggressiveness against the susceptible rice cultivar Galileo. Use of the EF‐1α sequence gave reliable identification and showed that Fusarium fujikuroi, the causal agent of bakanae disease, was the most abundant Fusarium spp. isolated. These data were confirmed by inoculation of the isolates to rice seeds which were then germinated in the greenhouse, showing that only F. fujikuroi isolates were able to cause bakanae disease. Pathogenic isolates were identified with different levels of aggressiveness. Phylogenetic analysis based on EF‐1α sequences generated a tree which separated the various Fusarium species into different clusters with high bootstrap values.     

Genetic variability and pathogenicity of Rhizoctonia solani associated with black scurf of potato in New Zealand

Isolates (a total of 129) of Rhizoctonia solani were collected from black scurf on potato tubers from different potato‐growing regions in New Zealand. Sequence analysis of the nuclear ribosomal DNA internal transcribed spacer (rDNA–ITS) regions from these isolates identified three anastomosis groups (AGs), AG‐3PT, AG‐2‐1 and AG‐5. Isolates classified as AG‐3PT were widely distributed, whereas AG‐2‐1 and AG‐5 were confined to distinct locations. Sequence heterogeneity was identified in the ITS regions of 100 AG‐3PT and AG‐2‐1 isolates. Variation in the sequence and length of the rDNA–IGS1 region was also observed for selected isolates of AG‐3PT and AG‐2‐1. Phylogenetic studies found all AG‐2‐1 isolates belong to AG‐2Nt, a subset of AG‐2‐1 previously associated with solanaceous crops in other countries. AG‐2‐1 isolates were consistently more aggressive than those of AG‐3PT. Delayed emergence, severe infection on stolons, formation of aerial tubers and considerable yield losses were associated with AG‐2‐1, but they caused negligible black scurf. In contrast, AG‐3PT caused black scurf on progeny tubers but variable effects on stem emergence and stolons. Furthermore, AG‐2‐1 isolates caused severe tuber malformation, but isolates of other AGs did not. This is the first report on the AG composition, genetic variability and pathogenicity of R. solani isolates associated with black scurf of New Zealand potatoes.     

Characteristics and pathogenicity of isolates of Rhizoctonia spp. associated with damping-off of sugar beet

Rhizoctonia solani and R. cerealis were isolated from diseased sugar-beet seedlings in Ireland. Isolates of R. solani were assigned to anastomosis groups AG-2, AG-4, AG-5 and an unidentified group that did not anastomose with recognized tester isolates. Cultures of AG-2 were similar to those of AG-5 on oatmeal agar (OA) and potato-dextrose-marmite agar (PDMA). Cultures of AG-4, the unidentified group and R. cerealis were morphologically distinct from one another, AG-2 and AG-5. The optimum temperature for growth of AG-2 was 225 C, with optimum growth of AG-4, AG-5 and the unidentified group at 275-C. R. cerealis grew slower than all groups of R. solani, with optimum growth at 225°C. Hyphae of R. cerealis were significantly narrower than those of the groups of R. solani studied. In glasshouse pathogenicity tests, some AG-2 and all AG-4, AG-5 and isolates from the unidentified group caused damping-off of beet seedlings. In controlled environments of 10-25°C, an AG-2 isolate was the most aggressive at 10 C whilst AG-4, AG-5 and the unidentified group caused most disease at or above 15°C. R. cerealis was also pathogenic to beet seedlings, causing damping-off at 10 and 15 C.     

Molecular characterization and pathogenicity of Pythium species associated with damping-off in greenhouse cucumber (Cucumis sativus) in Oman

A study was undertaken in 2004 and 2005 to characterize pathogens associated with damping-off of greenhouse-grown cucumber seedlings in 13 districts in Oman. Identification of Pythium to the species level was based on sequences of the internal transcribed spacer (ITS) of the ribosomal DNA. Of the 98 Pythium isolates collected during the survey, Pythium aphanidermatum , P. spinosum , P. splendens and P. oligandrum accounted for 76%, 22%, 1% and 1%, respectively. Pythium aphanidermatum was isolated from all of the districts, while P. spinosum was isolated from seven districts. Pathogenicity tests showed inter- and intraspecific variation in aggressiveness between Pythium species. Pythium aphanidermatum , P. spinosum and P. splendens were found to be highly aggressive at 25°C. However, the aggressiveness of P. spinosum decreased when the temperature was raised to 30°C, which was found to correspond to the lower frequency of isolation of P. spinosum in the warmer seasons, compared to the cooler time of the year. Pythium aphanidermatum exhibited limited intraspecific variation in the sequences of the ITS region of the rDNA and showed 100% similarity to the corresponding P. aphanidermatum sequences from GenBank. The ITS sequence data, as well as morphological characteristics of P. spinosum isolates, showed a high level of similarity within and between P. spinosum and P. kunmingense , and suggested that the two species were synonymous. This study represents the first report of P. spinosum, P. splendens and P. oligandrum in Oman.     

Effect of growth media, storage environment, soil temperature and delivery to soil on binucleate Rhizoctonia AG-G for protection of potato from Rhizoctonia canker

Binucleate Rhizoctonia (BNR) isolates propagated for 20 days at 24°C on oat kernels and for 30 days on vermiculite amended with potato broth were recovered from an average of 62% of whole kernels, 100% of chopped kernels and 71 % of vermiculite particles within the cultures, respectively. Viability of BNR isolates 232-CG and JF-3S4-3 was higher when stored at 5 than at 24°C, and was slightly affected by the vacuum used to reduce the O₂ level. After 17 weeks of storage at 5°C in air, BNR isolates 232-CG and JF-3S4-3 maintained similar viability (75% viability on whole oat kernels and 100% viability on chopped oat kernels), but in vermiculite amended with potato broth, viability of isolate 232-CG remained at 100% while that of JF-3S4-3 was 28%. In the glasshouse, BNR isolates 232-CG and JF-3S4-3 protected potato plants from Rhizoctonia canker caused by R. solani in soil maintained at 11, 17 and 23°C. Protection from Rhizoctonia canker was greater when BNR was delivered to soil than when placed on seed pieces. BNR-colonized-whole oat kernels placed in soil (15 g m of row) gave the greatest protection from Rhizoctonia canker in all experiments. In two field experiments in soil naturally infested with R. solani AG-3. the amount of BNR-colonized oat kernels was reduced from 15 g/m of row to 1-9 g m of row without affecting protection of potato plants from Rhizoctonia canker.     

Flooding causes loss in viability and pathogenicity of sclerotia of Rhizoctonia tuliparum

Samenvatting Rhizoctonia tuliparum veroorzaakt kwadegrond in tulp en iris. Sclerotiën van deze schimmel kunnen in grond zeer lang levensvatbaar en pathogeen blijven. Van sclerotiën van enkele schimmels is bekend, dat ze gevoelig zijn, voor inundatie die een aantal weken duurt. Inundatie wordt in Nederland vanaf 1982 door verscheidene, bollentelers 's zomers toegepast ter bestrijding van ziekten en onkruid.Sclerotiën vanR. tuliparum werden verpakt in nylon zakjes, ingegraven in emmers met grond en 1–6 weken geïnundeerd bij 17 °C. Op verschillende tijdstippen werden geïnundeerde en niet-geïnundeerde sclerotiën hetzij ontsmet, in 2,5% formaldehyde en vervolgens uitgeplaat op moutagar met oxytetracycline, hetzij aangebracht tussen de bruine huid en de buitenste bolrok van in kleine potjes geplante tulpebollen (cv. Apeldoorn); iedere bol werd geïnoculeerd met, een sclerotium. Na twee maanden werden de planten beoordeeld op symptomen van kwadegrond.In de eerste proef, waarbij sclerotiën werden uitgeplaat op agar, bleek er uit sommige sclerotiën die 2 weken waren geïnundeerd nog mycelium te groeien, terwijl dit na, 4 weken inundatie niet meer plaatsvond. In de tweede proef kiemden de sclerotiën op agar niet meer na 2 weken inundatie, maar meer dan de helft van de geïnoculeerde tulpebollen vertoonde wel symptomen. Pas als de sclerotiën 4 weken geïnundeerd waren bleken ze zodanig geïnactiveerd te zijn, dat er geen symptomen van kwadegrond werden waargenomen. Resultaten van veldproeven moeten nog worden afgewacht.     

Molecular tools to investigate Rhizoctonia solani distribution in soil

Real-time PCR protocols were developed to detect and discriminate 11 anastomosis groups (AGs) of Rhizoctonia solani using ribosomal internal transcribed spacer (ITS) regions (AG-1-IA, AG-1-IC, AG-2-1, AG-2-2, AG-4HGI+II, AG-4HGIII, AG-8) or β-tubulin (AG-3, AG-4HGII, AG-5 and AG-9) sequences. All real-time assays were target group specific, except AG-2-2, which showed a weak cross-reaction with AG-2^tabac. In addition, methods were developed for the high throughput extraction of DNA from soil and compost samples. The DNA extraction method was used with the AG-2-1 assay and shown to be quantitative with a detection threshold of 10⁻⁷ g of R. solani per g of soil. A similar DNA extraction efficiency was observed for samples from three contrasting soil types. The developed methods were then used to investigate the spatial distribution of R. solani AG-2-1 in field soils. Soil from shallow depths of a field planted with Brassica oleracea tested positive for R. solani AG-2-1 more frequently than soil collected from greater depths. Quantification of R. solani inoculum in field samples proved challenging due to low levels of inoculum in naturally occurring soils. The potential uses of real-time PCR and DNA extraction protocols to investigate the epidemiology of R. solani are discussed.     

禾谷丝核菌拮抗细菌的鉴定及其拮抗产物分析

 小麦纹枯病是长江中下游麦区重要的土传病害。明确小麦根际拮抗细菌种类和拮抗机理对小麦纹枯病生物防治具有重要意义。从江苏省姜堰市小麦根际分离到拮抗细菌A6、A12、B37、B40、B41和B42。通过生理生化测定和16SrDNA同源序列比对分析,这些菌株都为荧光假单胞杆菌。在温室条件下进行种子处理,这些菌株对小麦纹枯菌都有一定的控制作用,其中菌株A6、B41和B42的控病作用明显,菌株B42对小麦纹枯病的控制效果最好,温室防效达71.67%。基因检测和次生代谢物分析的结果表明:6个菌株都不产生2,4-二乙酰基间苯三酚和吩嗪,菌株A12、B37和B42含有与吡咯菌素合成相关的基因PrnD,在菌株B37中还检测到合成基因PrnC;菌株A12、B37和B42产生嗜铁素;除了菌株A6和B37,其余菌株都产生蛋白酶;所有菌株都不产生几丁质酶。     

云南香石竹立枯病病原的鉴定及致病性研究

 27 Rhizoctonia solani isolates from carnation with damping-off symptoms were identified as anastomosis group AG-4 based on cultural characteristics and anastomosis in Yunnan.Sequence analysis of 5.8S rDNA-ITS of yx and tx presented 99% sequence similarity with AG-4 HGⅠtester isolate.Pathogenicity tests showed that isolates of AG-4 HG Ⅰ were pathogenic to carnation.This is the first record of damping-off of carnation caused by AG-4 HG Ⅰ in China.