Assessment of resistance pathways induced in Arabidopsis thaliana by hypovirulent Rhizoctonia spp. isolates

Certain hypovirulent Rhizoctonia isolates effectively protect plants against well-known important pathogens among Rhizoctonia isolates as well as against other pathogens. The modes of action involved in this protection include resistance induced in plants by colonization with hypovirulent Rhizoctonia isolates. The qualifications of hypovirulent isolates (efficient protection, rapid growth, effective colonization of the plants, and easy application in the field) provide a significant potential for the development of a commercial microbial preparation for application as biological control agents. Understanding of the modes of action involved in protection is important for improving the various aspects of development and application of such preparations. The hypothesis of the present study is that resistance pathways such as systemic acquired resistance (SAR), induced systemic resistance (ISR), and phytoalexins are induced in plants colonized by the protective hypovirulent Rhizoctonia isolates and are involved in the protection of these plants against pathogenic Rhizoctonia. Changes in protection levels of Arabidopsis thaliana mutants defective in defense-related genes (npr1-1, npr1-2, ndr1-1, npr1-2/ndr1-1, cim6, wrky70.1, snc1, and pbs3-1) and colonized with the hypovirulent Rhizoctonia isolates compared with that of the wild type (wt) plants colonized with the same isolates confirmed the involvement of induced resistance in the protection of the plants against pathogenic Rhizoctonia spp., although protection levels of mutants constantly expressing SAR genes (snc1 and cim6) were lower than that of wt plants. Plant colonization by hypovirulent Rhizoctonia isolates induced elevated expression levels of the following genes: PR5 (SAR), PDF1.2, LOX2, LOX1, CORI3 (ISR), and PAD3 (phytoalexin production), which indicated that all of these pathways were induced in the hypovirulent-colonized plants. When SAR or ISR were induced separately in plants after application of the chemical inducers Bion and methyl jasmonate, respectively, only ISR activation resulted in a higher protection level against the pathogen, although the protection was minor. In conclusion, plant colonization with the protective hypovirulent Rhizoctonia isolates significantly induced genes involved in the SAR, ISR, and phytoalexin production pathways. In the studied system, SAR probably did not play a major role in the mode of protection against pathogenic Rhizoctonia spp.; however, it may play a more significant role in protection against other pathogens.     

Rhizoctonia spp. Recovered from Strawberry Roots in Central Coastal California

ABSTRACT Rhizoctonia spp. were commonly recovered from the roots of strawberry plants growing in nonfumigated soil in the central coastal region of California. With the exception of one multinucleate isolate of R. solani (frequency of recovery of 0.8%), all other isolates were binucleate and were in anastomosis groups (AG) A, G, or I. AGs-A and -I were recovered from all five collection sites, whereas AG-G was recovered from only two sites. AG-A was the most commonly isolated AG, followed by AGs-I and -G. Similar levels of virulence were observed among the different AGs, but differences in virulence were observed among isolates in the same AG. Evaluating anastomosis grouping by pairing isolates recovered from strawberry with known tester isolates did not always yield a positive anastomosis reaction, even though both isolates anastomosed with other members of the same AG. Subsequent investigations with multiple isolates in the same AG from the same collection location confirmed that there was a lack of anastomosis or weak anastomosis reactions for some combinations of pairings, highlighting the need for to use multiple tester isolates or molecular techniques for AG determination. Restriction fragment length polymorphism (RFLP) analysis of a polymerase chain reaction-amplified region of the rDNA was effective for differentiating AGs. Sixteen RFLP groups were observed after cluster analysis with data for the size of the amplified products and fragment sizes after digestion with four restriction enzymes. Although each AG had isolates in multiple RFLP groups, any one individual RFLP group contained isolates of only a single AG. There was no consistent correlation between RFLP group and location of isolate collection.     

Identification and Pathogenicity of Rhizoctonia spp. Isolated from Apple Roots and Orchard Soils

ABSTRACT Rhizoctonia spp. were isolated from the roots of apple trees and associated soil collected in orchards located near Moxee, Quincy, East Wenatchee, and Wenatchee, WA. The anastomosis groups (AGs) of Rhizoctonia spp. isolated from apple were determined by hyphal anastomosis with tester strains on 2% water agar and, where warranted, sequence analysis of the rDNA internal transcribed spacer region and restriction analysis of an amplified fragment from the 28S ribosomal RNA gene were used to corroborate these identifications. The dominant AG of R. solani isolated from the Moxee and East Wenatchee orchards were AG 5 and AG 6, respectively. Binucleate Rhizoctonia spp. were recovered from apple roots at three of four orchards surveyed and included isolates of AG-A, -G, -I, -J, and -Q. In artificial inoculations, isolates of R. solani AG 5 and AG 6 caused extensive root rot and death of 2- to 20-week-old apple transplants, providing evidence that isolates of R. solani AG 6 can be highly virulent and do not merely exist as saprophytes. The effect of binucleate Rhizoctonia spp. on growth of apple seedlings was isolate-dependent and ranged from growth enhancement to severe root rot. R. solani AG 5 and AG 6 were isolated from stunted trees, but not healthy trees, in an orchard near Moxee, WA, that exhibited severe symptoms of apple replant disease, suggesting that R. solani may have a role in this disease complex.     

水稻纹枯病生防内生菌糖蜜草固氮螺菌的分离与鉴定

 本文首先对砂仁内生细菌进行分离,以水稻纹枯病菌(Rhizoctonia solani)为靶标菌对获得菌株进行离体拮抗活性、盆栽及田间试验测定。结果表明：获得的27株内生细菌中有4株具有较好的离体抑菌活性,其中SRJ2-4抑菌效果最好,抑菌带达到18 mm;SRJ2-4的盆栽防效及田间防效分别为80.7%与79.4%,与其它菌株相比达极显著水平。SRJ2-4处理的亩产量为488.79 kg,高于其他药剂处理。对该菌株形态、生理生化及16S rDNA序列进行分析,将该内生菌鉴定为糖蜜草固氮螺菌(Azospirillum melinis)。     

Induction of systemic resistance by a hypovirulent Rhizoctonia solani isolate in tomato

A polynucleate Rhizoctonia isolate (R3) was analysed for virulence, growth characteristics, enzyme production and presence of dsRNAs. Taxonomic position was assessed morphologically and by anastomosis group (AG) testing and ITS sequence analysis. Results indicated that R3 is a hypovirulent R. solani AG 4. Mechanisms underlying biocontrol towards virulent R. solani and Botrytis cinerea were investigated and plant-mediated resistance was followed using biochemical markers of defence (PR1, laminarinase, chitinase). Control apparently relies on spatial and nutrient competition in soil, and on systemic induced resistance. This is the first report on induction of systemic resistance and of defence markers by a hypovirulent strain of R. solani.     

土壤产嗜铁素拮抗细菌CAS15的分离鉴定

采用CAS检测平板法,从海南岛橡胶树根际土壤分离到一株产嗜铁素能力较强的菌株CAS15,并通过室内平板对峙试验研究CAS15对15个常见病害病原菌的拮抗作用.结果显示,CAS15具有较强的拮抗作用,其菌液过滤液对病原菌抑制作用较强,抑制率为19.26%～94.07%.根据菌株形态特征、培养性状、生理生化特性及16S rDNA序列分析结果,将CAS15鉴定为枯草芽孢杆菌Bacillus subtilis.质谱分析和嗜铁素分析表明,CAS15产生的嗜铁素为儿茶酚型嗜铁素(DHB-Gly-Thr)3,分子质量为881.25,且嗜铁素的产生受到铁离子的抑制,当培养基中含有50μmol/L FeCl3时,其吸光度比值(OD510/OD600)为0.001～0.01.     

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.     

Genetic diversity of Meloidogyne spp. from rice and identification of multiresistant sources in Oryza spp. accessions

Recently a Meloidogyne species complex was detected parasitizing and causing damage to irrigated rice in southern Brazil, highlighting the need to study the genetic diversity of these species and their pathogenicity to Oryza spp. in order to select genotypes of rice with multiple resistance. This study compared the genetic diversity of Brazilian Meloidogyne spp. isolates from irrigated rice and evaluated the reaction of four wild accessions of Oryza species (O. glumaepatula, O. longistaminata, O. grandiglumis, and O. alta) and two cultivated species, O. glaberrima and O. sativa (control) to M. ottersoni, M. oryzae, and two variants of M. graminicola (Est G2 and Est G3). Genetic variability was assessed using RAPD and AFLP markers. M. graminicola and M. ottersoni showed high intraspecific variability: 83.76% and 41.14%, respectively. Cluster analysis showed a clear separation among rice root-knot nematodes (RKNs) into subclades according to their esterase phenotypes with 100% bootstrap. For rice resistance screening, plants were inoculated with 5,000 eggs, and the nematode reproduction factor evaluated 90–120 days postinoculation. O. glumaepatula, an American wild species, was highly resistant or resistant to all rice RKNs tested and is a valuable source of multiple resistance. Overall, the other rice species also showed different levels of resistance. Conversely, O. longistaminata exhibited low levels of resistance. M. graminicola Est G3 was the most aggressive isolate. Sources of resistance against RKN in wild Oryza genotypes, especially in an AA genome like O. glumaepatula, may be of great interest for future breeding programmes in cultivated rice.     

Nonpathogenic Binucleate Rhizoctonia spp. and Benzothiadiazole Protect Cotton Seedlings Against Rhizoctonia Damping-Off and Alternaria Leaf Spot in Cotton

ABSTRACT Recent reports have shown induction of resistance to Rhizoctonia root rot using nonpathogenic strains of binucleate Rhizoctonia spp. (np-BNR). This study evaluates the biocontrol ability of several np-BNR isolates against root and foliar diseases of cotton in greenhouse trials, provides evidence for induced systemic resistance (ISR) as a mechanism in this biocontrol, and compares the disease control provided by np-BNR with that provided by the chemical inducer benzothiadiazole (BTH). Pretreatment of cotton seedlings with np-BNR isolates provided good protection against pre- and post-emergence damping-off caused by a virulent strain of Rhizoctonia solani (AG-4). Seedling stand of protected cotton was significantly higher (P < 0.05) than that of nonprotected seedlings. Several np-BNR isolates significantly reduced disease severity. The combination of BTH and np-BNR provided significant protection against seedling rot and leaf spot in cotton; however, the degree of disease reduction was comparable to that obtained with np-BNR treatment alone. Significant reduction in leaf spot symptoms caused by Alternaria macrospora occurred on cotyledons pretreated with np-BNR or sprayed with BTH, and the np- BNR-treated seedlings had significantly less leaf spot than BTH-treated seedlings. The results demonstrate that np-BNR isolates can protect cotton from infections caused by both root and leaf pathogens and that disease control was superior to that observed with a chemical inducer.     

Enhanced molecular identification of Botrytis spp. from New Zealand onions

Botrytis spp. associated with neck rot disease were isolated from New Zealand onions. The fungi were identified using molecular sequences of the ribosomal internal transcribed spacer (ITS) and intergenic spacer (IGS) regions, and the glyceraldehyde-3-phosphate dehydrogenase (G3PDH) gene. Analyses of the sequences showed that the majority of the isolates gathered in 2005–07 were B. aclada. A new high resolution melting analysis (HRMA) assay was developed that allowed fast and simple discrimination between B. aclada and other Botrytis spp. causing onion neck rot in New Zealand. To further verify these results, Botrytis isolates from New Zealand onions, stored in the International Collection of Microorganisms from Plants (ICMP), were also examined. Only a single isolate from the ICMP collection was B. aclada while two isolates were B. byssoidea, one B. squamosa and another closely related to Botryotinia porri. Identification of the remaining Botrytis isolates was more difficult; while IGS and ITS sequences indicated a close relationship to B. allii or B. byssoidea, a previously unreported intron insertion was observed at the 3′ end of the ribosomal small subunit gene in these isolates. No evidence of heterogeneity was observed in the G3PDH gene sequences, as might have been expected of the allodiploid B. allii, but the G3PDH sequence ruled out B. byssoidea as the identity of these isolates.     

茄科蔬菜立枯丝核菌的融合群鉴定

 Sixty-five samples were collected from rhizosphere soil, hot pepper and tomato plants showing damping-off, root rot and stem rot in Taian, Shouguang of Shandong province and Zhouzhi, Taibai of Shaanxi province. Thirty-nine Rhizoctonia solani isolates were obtained from these samples. The results of anastomosis group (AG) identification and sequence analysis of 5.8S rDNA-ITS of the isolates showed that thirty-six isolates (92.3%) belonged to AG-4, while only three (7.7%) belonged to AG-5. The isolates of AG4 could further be divided into two subgroups of AG4-HG-Ⅰ and AG4-HG-Ⅲ. The 5.8S rDNA-ITS sequences of the selected isolates of the two subgroups had the 99%-100% identity with standard isolates of AG4-HG-Ⅰ and AG4-HG-Ⅲ (from GenBank). Among the analyzed isolates, AG4-HG-Ⅰ subgroup was the dominant with the frequency of 79.5%. Subgroup AG-4-HG-Ⅲ with the frequency of 12.8% was the second. This is the first report that subgroup AG4-HG-Ⅲ of R. solani isolated from Solanaceae vegetable crops in China.     

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.     

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.     

抑杀根结线虫的疣孢漆斑菌代谢活性物质的分离鉴定

正根结线虫病是设施蔬菜生产中一种常见病害(Chen et al.,2004),几乎能侵染温室内所有蔬菜,已严重影响该产业发展。真菌的胞外酶或者次生代谢毒素能促进寄生真菌对线虫的寄生(Fleming et al.,1990)或毒杀线虫(金娜等,2015)等。从高等真菌中筛选毒杀线虫的活性化合物,有助于获得新型、安全的高效杀线剂。疣孢漆斑菌Myrothecium verrucaria是生物防治寄生线虫的重要真菌,该菌产生的多种     

A selective medium for improving quantitative isolation ofTrichoderma spp. from soil

ATrichoderma-selective agar medium (TSM) was developed for quantitative isolation ofTrichoderma spp. from soil. Selectivity was obtained by using chloramphenicol as a bacterial inhibitor, and pentachloronitrobenzene, p-dimethylaminobenzenediazo sodium sulfonate and rose-bengal as selective fungal inhibitors. The TSM also contains a low concentration of glucose which still allows relatively rapid growth and sporulation ofTrichoderma, enabling convenient and rapid identification ofTrichoderma colonies. All the 15Trichoderma isolates tested formed colonies and grew well on this medium. Recovery ofTrichoderma from artificially inoculated soils was high and was not affected by soil type or by other microorganisms. A positive correlation was observed betweenTrichoderma added to soil and counts ofTrichoderma colonies on TSM plates. When combined with a soil pellet sampler, the selective medium was also used successfully for recovery of the indigenousTrichoderma population of natural soils.     

Isolation of mycoparasite Verticillium biguttatum from sclerotia of Rhizoctonia solani in the United Kingdom

Verticillium biguttatum was isolated from sclerotia of Rhizoctonia solani removed from black scurf-infected potato tubers which had been field-grown in the UK. Two isolates were identified by means of morphological and physiological characteristics, together with their ability to overgrow cultures of R. solani. A Dutch isolate M73 was used for comparison.     

A selective medium for isolation and identification ofBotrytis spp. from Soil and onion Seed

The population ofBotrytis allii in either naturally or artificially infested soils was selectively measured by the soil dilution plate count procedure on a developed synthetic medium supplemented with tannic acid, fungicides, antibiotics and Cu⁺⁺ions. Conversion of tannic acid into a dark brown pigment was related to the activity of extracellular polyphenoloxidase produced by the fungus. Thus, brown-pigmented colonies were recognized asBotrytis spp. The same medium was used for detecting the presence of the fungus on onion seed.     

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.     

Nematophagous Verticillium spp. in soils infested with Meloidogyne spp. in Cuba: Isolation and screening

Root-knot nematodes cause substantial economic loss of yield in coffee plantations and vegetable crops in Cuba. At present, methods to control the nematodes are ineffective or inappropriate and alternatives are being sought. The nematophagous fungus Verticillium chlamydosporium (Goddard) was isolated from soils collected from coffee plantations and infected root-knot nematode eggs from roots of tomato plants grown in these soils. A total of 83 isolates were collected and identified morphologically as V. chlamydosporium var. chlamydosporium, V. chlamydosporium var. catenulatum, V. psalliotae, V. suchlasporium and an isolate of V. chlamydosporium var. chlamydosporium with unusually large dictyochlamydospores. From these, 24 that represented a range of origins were selected and screened for their ability to parasitize eggs of root-knot nematodes, colonize the rhizosphere of barley roots and produce chlamydospores. None of the isolates grew at temperatures below 15°C and V. suchlasporium grew at a faster rate at lower temperatures than the other isolates. These were also screened in the glasshouse and V. chlamydosporium var. catenulatum caused the greatest reduction in nematode populations. One isolate of each subspecies of V. chlamydosporium was tested with the standard, Rothamsted isolate 10, on a range of host plants. The greatest reduction in numbers of nematodes occurred on tomato plants (cv. Pixie). The Rothamsted isolate 10 reduced numbers of nematodes toa greater extent than the other isolates, and therefore has the greatest potential as a biological control agent of root-knot nematodes.