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.     

Isolation and identification of hypovirulent Rhizoctonia spp. from soil

Two-hundred and forty-eight isolates of Rhizoctonia spp, were obtained from 13 locations in Gifu Prefecture in Japan using the plant debris particles isolation, colonization of bait tissue, and soil-clump plating methods. Of the isolates, 143 were binucleate Rhizoctonia spp., 60 were R. solani and 45 were R. zeae. Three isolates of R. solani and 54 of binucleate Rhizoctonia spp, were hypovirulent on radish, whilst all isolates of R. zeae were highly virulent, Hypovirulent strains were isolated most frequently by the plant debris particles isolation method, Hypovirulent isolates of R. solani belonged to anastomosis group 4, whilst the hypovirulent binucleate Rhizoctonia isolates belonged to AG A, AG Ba, AG G, and AG O.
Thirty-two isolates of Rhizoctotria spp, selected for hypovirulence on radish were tested on cucumber in vitro. Only five binucleate Rhizoctonia isolates and one R. solani isolate were hypovirulent on both species, and these isolates were also hypovirulent on seven other crop species. Cucumber showed wide variation in disease susceptibility to different isolates but hypovirulent isolates exhibited a consistent reaction on five different host cultivars, Pathogenicity tests using cucumber grown in soil also showed consistent reactions with isolates selected either for hypovirulence or virulence. The results support the use of cucumber in bioassays for identifying hypovirulent isolates of binucleate Rhizoctonia spp.     

Characterization, Genetic Structure, and Pathogenicity of Rhizoctonia spp. Associated with Rice Sheath Diseases in India

ABSTRACT Isolates of Rhizoctonia spp. were obtained from rice in India during 2000-2003. Characterization by conventional techniques and polymerase chain reaction showed that from 110 isolates, 99 were R. solani and 11 were R. oryzae-sativae. Of 99 isolates identified as R. solani, 96 were AG1-IA, 1 was AG1-IB, and 2 were AG1-IC. Amplified fragment length polymorphism (AFLP) analyzes were used to determine genetic relationships in Rhizoctonia pathogen populations collected from different geographic regions. Cluster analysis based on the AFLP data separated isolates belonging to the three different intraspecific groups of R. solani AG1 and differentiated R. solani from R. oryzae-sativae. Analysis of molecular variance (AMOVA) revealed that geographic region was the dominant factor determining population structure of R. solani AG1-1A; host cultivar had no significant effect. Pathogenicity tests on Oryza sativa cv. Zenith revealed that isolates of R. solani AG1-1A and AG1-1B were more virulent than R. solani AG1-IC and R. oryzae-sativae isolates.     

Identification and Quantification of Pathogenic Pythium spp. from Soils in Eastern Washington Using Real-Time Polymerase Chain Reaction

ABSTRACT Traditional methods of quantifying Pythium spp. rely on the use of selective media and dilution plating. However, high variability is inherent in this type of enumeration and counts may not be representative of the pathogenic population of Pythium spp. Variable regions of the internal transcribed spacer of the rDNA were used to design species-specific primers for detection and quantification of nine Pythium spp. from soils in eastern Washington. Primer pairs were designed for Pythium abappressorium, P. attrantheridium, P. heterothallicum, P. irregulare group I, P. irregulare group IV, P. paroecandrum, P. rostratifingens, P. sylvaticum, and P. ultimum and used with real-time polymerase chain reaction. Standard curves were generated for each of the species using SYBR Green I fluorescent dye for detection of amplification. Seventy-seven isolates of Pythium were screened to confirm specificity of each primer set. DNA was extracted from soil and standard curves were generated for P. irregulare group I, P. irregulare group IV, and P. ultimum to correlate populations of each species in the soil with quantities of DNA amplified from the same soil. Examination of raw field soils revealed results similar to those observed in previous studies. This new technique for the quantification of Pythium spp. is rapid and accurate, and will be a useful tool in the future study of these pathogenic Pythium spp.     

水花生病原菌——蕉斑镰刀菌菌株的筛选及其致病性测定

蕉斑镰刀菌 Fusarium stoveri是从自然界水花生植株上分离的、可致水花生表现萎蔫、地上茎腐烂、叶片上呈现褐色斑或黄化的生防真菌.室内研究表明,蕉斑镰刀菌菌丝生长和产孢的适宜温度范围在25～28℃,多数菌株的最适温度为28℃;菌株间的产孢量有较大差异.在供试菌株中,32-6菌株的产孢量最大;供试的9个蕉斑镰刀菌菌株对水花生均有较强的致病力,但菌株间的致病力有所差异.根据菌株的生长速率、产孢量和致病力等因素,菌株32-6被选为水花生生防菌的优选菌株.采用生产上常见农作物、蔬菜、经济作物及物候上与水花生一致的杂草等42种植物对菌株32-6进行寄主专化性测定,结果表明,蕉斑镰刀菌菌株32-6具有高度的寄主专化性,只对水花生表现出较强的致病性,对其他植物均不致病.     

Pathogenicity,host specificity and anastomosis groups ofRhizoctonia spp. isolated from soils in Israel

One hundred and twenty-nine isolates ofRhizoctonia spp. were obtained from soil samples in Israel and from culture collections in the U.S.A. and Japan. The isolates varied in host range and disease severity when tested on six to eleven different host plants. Approximately 30% of the isolates were nonpathogenic to all the host plants tested. Mycelial growth rate of the nonpathogenic strains did not differ significantly from that of the virulent isolates. The 107 Israeli isolates represented anastomosis groups (AG) 1, 2, 3, 4, 5 and 6 ofR, solani, two groups ofR. zeae, and three groups of binucleateRhizoctonia AG: A, F and K.     

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.     

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.     

苹果园瓢虫的助迁技术研究

在苹果蚜虫的防治关键期,间作冬油菜的苹果园中,冬油菜植株上瓢虫数量常远高于苹果树冠。本文通过研究发现,使用助迁技术能有效促使瓢虫从冬油菜向苹果树转移,能大量提高树冠上的瓢虫数量,增加对苹果蚜虫的自然控制力。     

Clarification of the Etiology of Glomerella Leaf Spot and Bitter Rot of Apple Caused by Colletotrichum spp. Based on Morphology and Genetic, Molecular, and Pathogenicity Tests

ABSTRACT Morphological characteristics and vegetative compatibility groups (VCGs) of 486 isolates of Glomerella cingulata, Colletotrichum gloeosporioides, and C. acutatum collected from apple leaves with Glomerella leaf spot (GLS) symptoms and fruit with bitter rot symptoms in the United States and Brazil were studied. From this collection, 155 isolates of G. cingulata (93 from fruit, 61 from leaves, and 1 from buds), 42 isolates of C. gloeosporioides from fruit, and 14 isolates of C. acutatum (10 from fruit and 4 from leaves) were studied using mitochondrial (mt)DNA restriction fragment length polymorphism (RFLP) haplotypes. A subset of 24 isolates was studied by examining the sequence of a 200-bp intron of the glyceraldehyde 3-phosphate dehydrogenase (GDPH) nuclear gene. In addition, 98 isolates were tested for pathogenicity on leaves of cvs. Gala and Golden Delicious in the greenhouse, and 24 isolates were tested for pathogenicity on fruit of cv. Gala in growth chambers. In total, 238 and 225 isolates of G. cingulata were separated into four distinct morphological types and six VCGs, respectively. Five morphological types and six VCGs were identified among 74 and 36 isolates of C. gloeosporioides, respectively. Three morphological types and four VCGs were identified among 74 and 23 isolates of C. acutatum, respectively. Seven different mtDNA RFLP haplotypes were observed within isolates of G. cingulata, two within isolates of C. gloeosporioides, and two within isolates of C. acutatum. Phylogenetic trees, inferred based on maximum likelihood and maximum parsimony methods using the intron sequence, produced similar topologies. Each species was separated into distinct groups. All isolates tested were pathogenic on fruit, though only isolates with specific VCGs and haplotypes were pathogenic to leaves. Vegetative compatibility was a better tool than molecular characters for distinguishing isolates of G. cingulata pathogenic on both leaves and fruit from the ones pathogenic only on fruit. Isolates of G. cingulata capable of causing both GLS and bitter rot were included in haplotypes and groups based on the sequence analysis of the 200-bp intron that also included isolates capable of causing bitter rot only. Additionally, isolates of G. cingulata from the United States and Brazil which cause GLS were included in different haplotypes and sequence analysis groups. Therefore, one hypothesis is that isolates of G. cingulata from the United States capable of causing both GLS on foliage and bitter rot on fruit may have arisen independently of Brazilian isolates of G. cingulata capable of causing both GLS and bitter rot, and the two groups of isolates may represent distinct populations.     

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.     

Protection of potato from Rhizoctonia canker with binucleate Rhizoctonia fungi

Fifteen isolates of binucleate Rhizoctonia fungi (BNR) were studied as potential biocontrol agents for protection of potato from Rhizoctonia canker in artificially infested greenhouse soil and potato fields naturally infested with Rhizoctonia solani (AG-3). Eight of the BNR reduced incidence and severity of Rhizoctonia stem canker in greenhouse experiments by an average of 78 and 85%, respectively. In a field naturally infested with R. solani, selected isolates of BNR and the fungicide Tops 2.5D (thiophanate-methyl) were equally protective of potato from Rhizoctonia stem canker. BNR isolates gave protection of potato from Rhizoctonia stolon canker similar to PCNB and superior to Tops 2.5D. Cultivars Atlantic, Irish Cobbler, Kennebec, Norchip, Russet Burbank, and Superior were protected equally from Rhizoctonia stem canker by selected isolates of BNR under field conditions. Isolates of BNR show potential as biocontrol agents for protection of potato from Rhizoctonia canker.     

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.     

Detection of Phytophthora nicotianae and P. citrophthora in Citrus Roots and Soils by Nested PCR

The polymerase chain reaction (PCR) was used for the specific detection of Phytophthora nicotianae and P. citrophthora in citrus roots and soils. Primers were based on the nucleotide sequences of the internal transcribed space regions (ITS1 and ITS2) of 16 different species of Phytophthora. Two primer pairs, Pn5B–Pn6 and Pc2B–Pc7, were designed specifically to amplify DNA from P. nicotianae and P. citrophthora, respectively. Another primer pair (Ph2–ITS4) was designed to amplify DNA from many Phytophthora species. All primer pairs were assessed for specificity and absence of cross-reactivity, using DNA from 118 isolates of Phytophthora and 82 of other common soil fungi. In conventional PCR, with a 10-fold dilution series of template DNA, the limit of detection was of 1pgl^–1 DNA for all the primer pairs (Ph2–ITS4, Pn5B–Pn6, and Pc2B–Pc7). In nested PCR, with primers Ph2–ITS4 in the first round, the detection limit was of 1fgl^–1 for both the primer sets (Pn5B–Pn6 and Pc2B–Pc7). Simple, inexpensive and rapid procedures for direct extraction of DNA from soil and roots were developed. The method yielded DNA of a purity and quality suitable for PCR within 2–3h. DNA extracted from soil and roots was amplified by nested PCR utilizing primers Ph2–ITS4 in the first round. In the second round the primer pairs Pn5B–Pn6 and Pc2B–Pc7 were utilized to detect P. nicotianae and P. citrophthora, respectively. Comparison between the molecular method and pathogen isolation by means of a selective medium did not show any significant differences in sensitivity.     

Identification of a uninucleate Rhizoctonia sp. by pathogenicity, hyphal anastomosis and RAPD analysis

Finnish and Norwegian uninucleate Rhizoctonia sp, isolates. originating from roots of nursery grown conifer seedlings suffering from root dieback, and having Ceratobasidium perfect state, were tested for pathogenicity and genetic related ness. All tested isolates of this pathogen considerably reduced the root system development of Scots pine and Norway spruce seedlings resulting in death or stunted growth. The uninucleate isolates anastomosed readily with each other producing a killing reaction. In a RAPD-PCR analysis, the uninucleate isolates had different banding patterns from our reference isolates, two Finnish binucleate isolates (AG-I and R. sp.) and standard tester isolates of genus Ceratobasidium representing anastomosis groups AG-A, AG-C, AG-E, AG-G and AG-I. UPGMA analysis clustered the uninucleate isolates together at a greater similarity than 75% while the binucleate isolates formed distinct clusters and were 10-25% similar to the uninucleate Rhizoctonia sp. Hyphal anastomosis and DNA data suggest that the uninucleate Rhizocionia sp. is an homogeneous group and distinct from the tested binucleate Rhizoctonia isolates.     

陕西苹果园土壤砷和重金属污染评价

应用地积累指数和生态危害指数,对陕西苹果主产区果园土壤砷(As)和重金属污染进行了评价。结果表明:从应用黄土母质土壤背景值计算的平均地积累指数结果来看,果园土壤中Hg达到轻-中度污染,Cd,Cr,Pb,Cu,As 5种元素都没有造成污染。从6种元素的地积累指数分级频率来看,Hg和As的污染频率最大,达到46.8%;其次是Cd和Cu,分别是27.4%和4.8%;Pb和Cr均没有造成污染。从6种元素的潜在生态危害系数来看,果园土壤中Hg的生态风险危害最大,其次为Cd,其余4种元素都为轻微危害程度,生态危害系数均小于20;从果园土壤As和重金属的生态危害指数来看,有27.42%的果园达轻微危害,56.45%达中等危害,16.13%达强危害程度。     

Identification, pathogenicity and distribution of Penicillium spp. isolated from garlic in two regions in Argentina

A total of 147 samples of garlic ( Allium sativum ) bulbs affected by blue mould were obtained from a variety of agroclimatic districts between December 1999 and February 2000. Penicillium species were identified using both morphological and chemotaxonomic characteristics. Penicillium allii was the predominant species isolated (81·8%) in this survey and the only species proven to be pathogenic on garlic. Other species were isolated much less frequently: P. chrysogenum (13·7%), P. brevicompactum (2·8%), P. phoeniceum (0·9%), P. aurantiogriseum (0·6%) and P. flavigenum (0·2%). Colonies of P. allii could be classified into four morphotypes and their distribution seemed to be influenced by seed trade and agricultural practices. Penicillium allii isolates were grouped into three aggressiveness phenotypes (low, medium and high) based on their ability to cause disease during field trials on susceptible (Fuego INTA) and less susceptible (Castaño INTA) garlic cultivars. The number of surviving plants at 191 days after planting and postharvest bulb weight contributed the most towards aggressiveness modelling.     

Anastomosis Groups, Pathogenicity and Sensitivity to Fungicides of Rhizoctonia solani Isolates Collected on Potato Crops in France

A collection of 241 isolates of Rhizoctonia solani obtained from potato plants grown in different areas in France was characterized for anastomosis grouping, symptomatology on tubers of different cultivars and sensitivity to three fungicides. Most isolates collected belonged to (anastomosis groups (AGs)) AG 3, but 2% and 4% of the isolates were AG 5 and AG 2-1. AG 3 and AG 2-1 isolates were mostly obtained from sclerotia on tubers, but all AG 5, some AG 3 and some AG 2-1 isolates were recovered from superficial tuber alterations, like deformations, corky or scabby lesions. Sclerotia were formed on tubers produced by healthy stem cuttings grown in soil artificially infested with AG 3, but not on tubers grown in soil infested with either AG 5 or AG 2-1. No variation in susceptibility to sclerotial formation was observed among five potato cultivars. In all cases, a large proportion of tubers showed superficial corky lesions, often associated with deformations. The proportion of tubers with lesions and deformations was highest in soil infested with AG 2-1 and significantly lower on cv. Samba in all treatments. All isolates were highly sensitive to flutolanil, iprodione and pencycuron, except the AG 5 isolates, moderately sensitive to pencycuron. These results show that, although AG 3 is the most common R. solani group on potato in France, AG 5 and AG 2-1 may be present. Isolates differed for pathogenicity. In vitro sensitivity to fungicides varied among AGs.