Tester Isolates Research Articles

Fertility and Mating Type Frequency in Indian Isolates of Sclerospora graminicola, the Downy Mildew Pathogen of Pearl Millet.

Sclerospora graminicola, the downy mildew pathogen of pearl millet, is an oomycetous obligate parasite which reproduces by both sexual and asexual means. Fertility and mating type frequencies were studied in 70 single-zoospore isolates (SZIs) obtained from seven representative oo-sporic isolates (Sg 021, Sg 048, Sg 110, Sg 139, Sg 149, Sg 152, and Sg 153) of S. graminicola collected from major pearl millet-growing states of India. Of the 70 SZIs tested for fertility according to oospore production potential, 62 were self-sterile and 8 were self-fertile, indicating the low occurrence of homothallism in the S. graminicola populations. The sexual mating type test of the 70 SZIs, conducted by pairing each isolate with the two standard mating type tester isolates PT2 (Mat A) and PT 3 (Mat B), revealed 28 (40.0%) isolates of Mat A, 33 (47.14%) of Mat B, 8 (11.43%) of both Mat A and Mat B, and 1 (1.43%) as unknown. The frequencies of Mat A and Mat B were in approximately equal proportions among the isolates tested, except in three parental isolates. Implications of these results in understanding the dynamic genetic structure of S. graminicola population and potential for evolution of new virulence in the pathogen are discussed.

Molecular‐based identification and phylogeny of Armillaria species from Serbia and Montenegro

Summary Armillaria causes problems of root rot, kill trees and decay wood in the forests of Serbia and Montenegro, but the species involved have not hitherto been identified. The aim of this study was to identify field isolates collected on 25 localities. Identification was based on restriction fragment length polymorphism (RFLP) analysis of intergenic spacer 1 (IGS1) region and comparisons of IGS1 sequence with those available on NCBI database. Phylogenetic analysis was performed on sequence information from selected isolates to determine possible interrelationships between isolates with different banding patterns and previously identified tester isolates of five European Armillaria species. Five Armillaria species were identified in 90 isolates obtained from forests in Serbia and Montenegro. Armillaria gallica was most frequently isolated, followed by A. cepistipes, A. mellea, A. ostoyae and A. tabescens; two isolates remained unidentified. Restriction digestion of IGS1 amplification products with AluI produced 10 RFLP patterns. Patterns G4 (400, 250, 180) for A. gallica and pattern X (400, 180, 140) for isolates 74 and 79 are reported for the first time in European isolates. Eight RFLP patterns were observed after restriction with TaqI. Two patterns each were observed for A. ostoyae and A. gallica, and one each for A. cepistipes, A. mellea, A. tabescens and isolates 74 and 79. Parsimony analyses based on the IGS1 region placed the isolates into four clades: one including A. mellea, the second containing A. gallica–A. cepistipes isolates, while isolates of A. ostoyae and A. borealis were in the third clade. Armillaria tabescens differed from all annulate species. Phylogenetic analysis supported the conclusion that European Armillaria species are closely related and separated from a common ancestor in the near past. According to this survey five European Armillaria species are present in the forests of Serbia and Montenegro, while A. borealis is not present in the studied ecosystems.

Distribution of Mating Types and Diversity in Virulence of Didymella rabiei in Israel

The distribution of mating types and diversity in virulence of Didymella rabiei populations were studied in Israel from 1997 to 1999. Forty-one monoconidial D. rabiei isolates from 18 commercial fields distributed among all the chickpea production areas of the country were paired with MAT1-1 and MAT1-2 mating type tester isolates of D. rabiei. Both mating types were found in all chickpea production areas of the country. Of the 18 fields sampled, MAT1-1 was observed in 44%, and MAT1-2 in 88% of the sites. In some sites both mating types were present in close proximity, suggesting that sexual reproduction of the pathogen was feasible. The contribution of sexual reproduction of the fungus to virulence diversity was tested on detached leaves of six differential chickpea cultivars. Nine isolates were derived from different well separated foci (derived from ascospores as inoculum) and eight isolates were derived from a single, well defined infection focus (derived from sister conidia). In the analyses of variance the cultivar × isolate interaction showed no significant (P of F>0.09) effect on disease incidence; the chickpea cultivars differed significantly (P of F 0.1) among isolates of the ascosporic population. Nevertheless, when comparing a cultivar at a time, the ascosporic and conidial populations did not differ significantly (P of F>0.1) in their virulence diversity. Virulence of 41 isolates collected from the different chickpea fields was tested on detached leaves of four Israeli cultivars that differ in their field response to D. rabiei. The cultivar × isolate interaction showed no significant effect (P of F = 0.95) on disease incidence. The main effects of cultivar and isolate on disease incidence were highly significant (P of F<0.0001). Accordingly, our data do not support the hypothesis that there is pathogenic specialization in the D. rabiei–C. arietinum pathosystem in Israel.

Identification of an Avirulence Gene in the Fungus Magnaporthe grisea Corresponding to a Resistance Gene at the Pik Locus

ABSTRACT A rice isolate of Magnaporthe grisea collected from China was avirulent on rice cvs. Hattan 3 and 13 other Japanese rice cultivars. The rice cv. Hattan 3 is susceptible to almost all Japanese blast fungus isolates from rice. The genetic basis of avirulence in the Chinese isolate on Japanese rice cultivars was studied using a cross between the Chinese isolate and a laboratory isolate. The segregation of avirulence or virulence was studied in 185 progeny from the cross, and monogenic control was demonstrated for avirulence to the 14 rice cultivars. The resistance gene that corresponds to the avirulence gene (Avr-Hattan 3) is thought to be located at the Pik locus. Resistance and susceptibility in response to the Chinese isolate in F(3) lines of a cross of resistant and susceptible rice cultivars were very similar to the Pik tester isolate, Ken54-20. Random amplified polymorphic DNA markers and restriction fragment length polymorphism markers from genetic maps of the fungus were used to construct a partial genetic map of Avr-Hattan 3. We obtained several flanking markers and one co-segregated marker of Avr-Hattan 3 in the 144 mapping population.

First Report of AG-A of Binucleate Rhizoctonia in China, Pathogenic to Soya Bean, Pea, Snap Bean and Pak Choy

Twenty binucleate Rhizoctonia (BNR) isolates were collected from roots of soya bean, pea, snap bean and pak choy with root rot symptoms in Yunnan Province, China. Chinese isolates anastomosed with the tester isolate of anastomosis group-A (AG-A; C-517) with a high C2 fusion rate (>70%). Chinese isolates were pathogenic to soya bean, pea, snap bean and pak choy and had 97% similarity sequence of 5.8S rDNA-internal transcribed spacer with AG-A tester isolates SN-2 and C-662. When compared with other groups, AG-Ba and AG-Bb, Chinese isolates showed 77% sequence similarity. These results show that Chinese isolates belong to AG-A of BNR. Growth rate, hyphal diameter, cultural characteristics and pathogenicity of the Chinese isolates differed significantly from the tester isolate of AG-A. This is the first report on AG-A in China.

Assessment ofMagnaporthe griseamating type by PCR

AbstractAnalysis of mating type can provide an evaluation of the population genetic diversity of the rice blast fungus,Magnaporthe grisea. According to the sequences ofMAT1-1andMAT1-2genes of the fungus, two pairs of PCR primers specific to theMAT1-1andMAT1-2alleles were designed, and the PCR thermal profile was also optimized. To confirm its application in mating type assessment, 10 tester isolates were tested by PCR. The PCR amplification pattern of these tester isolates corresponded to their known mating type. Furthermore, 150 rice-field isolates from Fujian Province were mated with tester isolates GUY11 and KA3 side by side and also tested by PCR. Results showed that 95.1% of 123 fertile isolates were the same in mating type as determined by both PCR-amplified allele-specific fragments and mating with GUY11/KA3. Among 27 sterile isolates determined by GUY11 and KA3, seven wereMAT1-1and 20 wereMAT1-2as determined by PCR. This study indicates that PCR is applicable in assessingM. griseamating type and especially is capable of predicting the potential mating type of sterile isolates in the natural population of the fungus.

Vegetative compatibility groups inVerticillium dahliae isolates from cotton in Turkey

Verticillium dahliae Kleb. with a complicated genetic diversity is a widely distributed major pathogen resulting in cotton wilt, which causes high economic losses in cotton lint production in the cotton belt of Turkey. A collection of 70 TurkishV. dahliae isolates (68 from wilted cotton plants in 28 districts and two from watermelon plants in two districts) were tested for vegetative compatibility by observing heterokaryon formation among complementary nitrate-nonutilizing (nit) mutants. The mutants were tested against international reference tester isolates and also were paired with one another. Thirty-nine isolates were assigned to vegetative compatibility group (VCG) 2B, 19 to VCG2A and three to VCG4B. One isolate was self-incompatible and eight others could not be assigned to any of the identified VCGs because theirnit mutants showed negative reactions with the tester isolates of four VCGs or theirnit mutants reverted back to the wild type. This is the first report of VCGs inV. dahliae from cotton in Turkey.

Characterization of Rhizoctonia solani AG 4HG-III Causing Stem Canker and Wirestem on Green Amaranth and Chinese Amaranth

During December 2003, stem canker and wirestem were observed on the stems of green amaranth (Amaranthus viridis) and Chinese amaranth (Amaranthus tricolor) in greenhouses at Ximao district in Yunnnan Province, China. Isolates of Rhizoctonia solani obtained from the two amaranths with stem canker and wirestem, were identical to anastomosis group (AG)-4. The isolates from diseased plant showed high virulence on young seedlings of two amaranths. Results of sequence analysis of 5.8s rDNA-ITS of Chinese isolates showed 99-100% sequence similarity with AG-4HG-III tester isolates. When compared with other subgroups of AG-4, Chinese isolates showed similarity levels of 94%. This is the first report of stem canker and wirestem of Green amaranth and Chinese amaranth caused by AG-4HG-III and AG-4HG-III in China.

First Report of Root Rot of Bean and Soybean Caused by Rhizoctonia zeae in Turkey.

To determine the species of Rhizoctonia on bean and soybean plants grown in Samsun (Turkey), field surveys were performed at 104 locations during 2001 and 2002. Rhizoctonia spp. were obtained from isolations from the necrotic lesions on the hypocotyl and rhizosphere soils. Species were identified according to Ogoshi (3) on the basis of hyphal and colony morphology and anastomosis reaction with known tester isolates (provided by M. Hyakumachi, Gifu University, Japan). Fifty Rhizoctonia spp. isolates obtained from these locations were identified as Rhizoctonia zeae (teleomorph Waitea circinata var. zeae). Nine of the 27 bean isolates and 8 of the 23 soybean isolates were recovered from plant tissues. These isolates had optimum temperature (32°C) for growth. Colonies were orange when young, becoming salmon colored with age. Sclerotia formed both on the agar surface or submerged in the medium. Superficial sclerotia were more uniform and nearly spherical, mostly 0.2 to 0.5 mm in diameter, and they were first orange and then turned brown. Pathogenicity was tested with three R. zeae isolates grown on sterile oat seeds at 25°C for 10 days. Bean and soybean seedlings grown in 1-liter plastic pots containing sterile potting mix (field soil/composted manure/sand 2:2:1 [v/v]) at true-leaf stage were inoculated by placing five infested oat seeds adjacent to the roots. Sterile oat seeds were used for controls. After 3 to 4 weeks of incubation at 17 to 25°C in a glasshouse, roots of the plants were cleaned with tap water and evaluated for disease severity. Four replicate pots were used for each isolate/plant combination. All isolates produced superficial brown lesions on roots and hypocotyls similar to those observed on plants used for isolations and root growth declined. R. zeae was reisolated from the lesions on all bean and soybean plants used for the pathogenicity test. While R. zeae was previously reported from Johnsongrass roots (1) and corn kernels (2), to our knowledge, this is the first report of R. zeae isolated from bean and soybean plants and rhizosphere soils in Turkey.

First Report of Summer Patch of Creeping Bentgrass Caused by Magnaporthe poae in North Carolina.

An unknown disease was observed in June 2002 and 2003 on creeping bentgrass (CRB [Agrostis stolonifera L.]) putting greens at The Country Club of Landfall in Wilmington, NC that were established in 2001 with a 1:1 blend of cvs. A-1 and A-4. Soil pH ranged from 7 to 8 at this location because of poor quality irrigation water. Symptoms appeared in circular patches of 0.3 to 1 m in diameter that exhibited signs of wilt followed by chlorosis and orange foliar dieback. The disease was initially diagnosed as take-all patch caused by Gaeumannomyces graminis (Sacc.) Arx & D. Olivier var. avenae (E.M. Turner) Dennis, based on the observation of necrotic roots and crowns that were colonized with dark, ectotrophic hyphae. However, the historical lack of take-all patch occurrence in this region led to the suspicion that G. graminis var. avenae was not involved. Sections of root and crown tissue were surface disinfested in 0.6% NaOCl for 5 min or 1% AgNO3 for 1 min and 5% NaCl for 30 s. Tissue was plated on SMGGT3 (2) or on potato dextrose agar containing 50 mg L-1 of tetracycline, streptomycin, and chloramphenicol. A fungus resembling Magnaporthe poae Landschoot & Jackson was consistently obtained regardless of isolation method. Teleomorph production was conducted on Sachs agar (4) overlaid with autoclaved wheat (Triticum aestivum L.) stem sections. Seven isolates were plated alone or paired with M. poae tester isolates 73-1 or 73-15 (3) and incubated at room temperature under continuous fluorescent illumination. Six isolates produced perithecia and ascospores typical of M. poae (3) when paired with 73-15 but not when plated alone or paired with 73-1; these isolates are, therefore, M. poae mating type 'a'. Isolate TAP42 did not produce perithecia and remains unidentified. Cone-Tainers (3.8 × 20 cm) containing calcined clay were seeded with 'A-4' CRB (9.7 g cm-2) and inoculated 8 weeks later by placing four M. poae-infested rye (Secale cereale L.) grains below the soil surface. Inoculated Cone-Tainers were placed in growth chambers with 12-h day/night cycles at 30/25°C, 35/25°C, or 40/25°C. Field plots (1 m2) of 'A-4' CRB in Jackson Springs, NC were inoculated on 19 June 2003 by removing a soil core (1.9 × 10.3 cm) from the center of each plot, adding 25 cm3 of M. poae-infested rye grains, and then capping the hole with sand. Growth chamber and field inoculations were arranged in a randomized complete block with four replications. Eight weeks after inoculation in the growth chamber, isolates TAP35, TAP41, and SCR4 caused significant foliar chlorosis and dieback at 12-h day/night cycles of 30/25°C and 35/25°C, but only TAP41 induced symptoms at 40/25°C. Isolate TAP42 did not induce symptoms at any temperature regimen. Orange patches (10 to 15 cm in diameter) were observed in field plots inoculated with TAP41 on 27 August 2003. No other isolates induced aboveground symptoms. Roots and crowns of plants exhibiting foliar symptoms in the greenhouse and field were necrotic and colonized with ectotrophic hyphae, and M. poae was consistently isolated from this tissue. Although M. poae has been associated with CRB in Florida (1), to our knowledge, this is the first report of summer patch of CRB within the normal zone of adaptation for this turfgrass species. Observation of this disease highlights the need for accurate methods for diagnosis of diseases caused by ectotrophic root-infecting fungi.

First report of Rhizoctonia solani on Diplotaxis tenuifolia in Italy

Recently, perennial wallrocket (Diplotaxis tenuifolia) has become a widespread crop in the Piana del Sele area, Campania, southern Italy. In 2001, wallrocket was cropped on about 500 ha under glass (Giorgini, 2001) and by 2003 the area under cultivation had increased further. In the past 3 years, bare patches have appeared on a number of farms in the area. Typically, germination failures and damping-off of plantlets were observed at the centre of the patch, while plants at the border showed slower development, yellowing and early senescence of leaves. Necrotic lesions developing on one side of both crown and taproot were also evident. Isolations from infected tissues carried out on 2% water agar (WA) yielded 27 multinucleate Rhizoctonia isolates from samples collected in 12 different locations. Pairings on WA were made with tester isolates belonging to anastomosis groups (AG) 1 through to 11 of R. solani (according to Carling, 1996). All the isolates anastomosed with high fusion frequency, with tester isolates Rh-74 and RT32 belonging to AG-4. Pathogenicity was tested in plastic pots containing sterilized soil infested with three selected isolates. Most seedlings underwent damping-off in a few days, while the remaining showed more or less developed necrotic areas at the crown level. Uninoculated control plants were unaffected. There are no previous reports of R. solani on perennial wallrocket. Although R. solani is a common pathogen on Brassicaceae, most reports in the literature, however, refer to AG-2-1. AG-4 is known as the most polyphagous anastomosis group and it is believed to be able to infect plantlets of any crop species at the soil line (Anderson, 1982). Therefore, the increasing incidence of Rhizoctonia on perennial wallrocket appears to be related to an obvious increase of its inoculum potential in the cultivated soil caused by the repeated cropping of this plant species and to the persistence in the soil of the crop residues after the final harvest.

Genetic Diversity of Sclerotinia homoeocarpa Isolates from Turfgrasses from Various Regions in North America.

Sixty-seven isolates of Sclerotinia homoeocarpa, causing dollar spot disease in creeping bentgrass, annual bluegrass, Bermudagrass, and perennial ryegrass turf, collected from 23 golf courses in various geographical regions of the United States and Canada between 1972 and 2001, were characterized by vegetative compatibility, genetic diversity, and pathogenicity. Eleven vegetative compatibility groups (VCGs A to K) were identified among the isolates tested in this study, and five of them (VCGs G to K) were new. VCG B was the most predominant group, typifying 33 isolates (51%) tested. S. homoeocarpa isolates collected from golf courses in Pennsylvania belonged to seven VCGs (A, B, E, F, G, I, and K), whereas three groups were observed in those collected from New York (B, E, and G) and New Jersey (E, H, and I). Two isolates, one each from Pennsylvania and Canada, were incompatible when paired with the tester isolates in all possible combinations, and did not fall into any known VCG. An isolate collected from Canada was compatible with tester isolates from two VCGs (C and D). Genetic analyses using amplified fragment length polymorphism (AFLP) showed the presence of two genetically distinct groups, designated as major group and the minor group. The major group included 36 isolates collected from various golf courses in the United States and Canada. Two isolates collected from bermudagrass in Florida formed a separate cluster, the minor group. Isolates that belonged to the major group were further divided into two subgroups (1 and 2). Subgroup 1 consisted of all the isolates that belonged to VCGs A, E, G, H, and I. Three of the four isolates that belonged to VCG K also were clustered with isolates of subgroup 1. Subgroup 2 consisted of all the isolates from VCG B, and one each from VCGs F and K. Pathogenicity assays on Penncross creeping bentgrass showed significant differences (P = 0.05) in virulence among the isolates. Overall, a relationship between virulence and VCGs was observed, in which certain virulence groups corresponded to specific VCGs; however, such a relationship was not observed between virulence and AFLPs. Close similarity among isolates of S. homoeocarpa collected from different locations in the United States and Canada suggests that isolates of the same genotype could be involved in outbreaks of dollar spot epidemics at multiple locations.

Molecular Mapping of the Stb4 Gene for Resistance to Septoria tritici Blotch in Wheat

ABSTRACT Breeding wheat for resistance is the most effective means to control Septoria tritici blotch (STB), caused by the ascomycete Mycosphaerella graminicola (anamorph Septoria tritici). At least eight genes that confer resistance to STB in wheat have been identified. Among them, the Stb4 locus from the wheat cv. Tadinia showed resistance to M. graminicola at both seedling and adult-plant stages. However, no attempt has been made to map the Stb4 locus in the wheat genome. A mapping population of 77 F10 recombinant-inbred lines (RILs) derived from a three-way cross between the resistant cv. Tadinia and the susceptible parent (Yecora Rojo x UC554) was evaluated for disease resistance and molecular mapping. The RILs were tested with Argentina isolate I 89 of M. graminicola for one greenhouse season in Brazil during 1999, with an isolate from Brazil (IPBr1) for one field season in Piracicaba (Brazil) during 2000, and with Indiana tester isolate IN95-Lafayette-1196-WW-1-4 in the greenhouse during 2000 and 2001. The ratio of resistant:susceptible RILs was 1:1 in all three tests, confirming the single-gene model for control of resistance to STB in Tadinia. However, the patterns of resistance and susceptibility were different between the Indiana isolate and those from South America. For example, the ratio of RILs resistant to both the Indiana and Argentina isolates, resistant to one but susceptible to the other, and susceptible to both isolates was approximately 1:1:1:1, indicating that Tadinia may contain at least two genes for resistance to STB. A similar pattern was observed between the Indiana and Brazil isolates. The gene identified with the Indiana tester isolate was assumed to be the same as Stb4, whereas that revealed by the South American isolates may be new. Bulked-segregant analysis was used to identify amplified fragment length polymorphism (AFLP) and microsatellite markers linked to the presumed Stb4 gene. The AFLP marker EcoRI-ACTG/MseI-CAAA5 and microsatellite Xgwm111 were closely linked to the Stb4 locus in coupling at distances of 2.1 and 0.7 centimorgans (cM), respectively. A flanking marker, AFLP EAGG/ M-CAT10, was 4 cM from Stb4. The Stb4 gene was in a potential supercluster of resistance genes near the centromere on the short arm of wheat chromosome 7D that also contained Stb5 plus five previously identified genes for resistance to Russian wheat aphid. The microsatellite marker Xgwm111 identified in this study may be useful for facilitating the transfer of Stb4 into improved cultivars of wheat.

Rhizoctonia solani AG-13 Isolated from Corn in Mississippi.

Rhizoctonia solani K¨hn anastomosis group (AG) 13 was isolated from asymptomatic root tissue of a corn (Zea mays L.) seedling grown at the Black Belt Branch Experiment Station, Brooksville, MS. Rhizoctonia solani AG-13 was recently reported from cotton grown in Georgia (2). Rhizoctonia solani isolate MS-168 was successfully anastomosed with tester isolate AG-13 (courtesy of D. E. Carling, University of Alaska). The hyphal diameter at the point of anastomosis was reduced, the anastomosis point was obvious, and cell death of adjacent cells was observed. These results were confirmed by D. E. Carling and are consistent with C2 anastomosis hyphal reactions (1). Rhizoctonia solani isolate MS-168 was cultured on potato dextrose agar (PDA) and incubated at 27 ± °C with a 12-h photoperiod for 28 days. Mycelium was buff-brown to beige with diurnal zonation throughout the colony. Aggregates of bulbils developed in the center of the colony that were dark brown, dome shaped, and accompanied by brown exudate. Bulbils were submerged in the medium and scattered across the surface of the colony. The optimal growing temperature of MS-168 was 27°C. Two pathogenicity evaluations were conducted on 170 corn seedlings planted into soilless potting medium. Four-day-old corn seedlings were inoculated with 7-day-old PDA hyphal plugs (2 mm in diameter) of R. solani isolate MS-168 by placing the mycelium side of the hyphal plug in contact with the mesocotyl tissue beneath the soil surface. The hyphal plugs were covered with soil. The nontreated corn seedlings were inoculated with PDA plugs minus the fungus. Corn seedlings were maintained under environmentally controlled conditions at 27 ±2 °C with a 12-h photoperiod and watered to prevent wilting. Disease symptoms on mesocotyl tissue were rated from 1 to 4 in which 1 = no symptoms, 2 = a few pinpoint lesions and diffuse discolored areas, 3 = distinct necrotic lesions, and 4 = girdling lesions (3). Fourteen days postinoculation, treated seedlings had a significantly higher disease rating (1.5) than the nontreated control (1.0). Thirty of eighty-seven corn seedlings inoculated with MS-168 expressed symptoms of discoloration and pinpoint and necrotic lesions on the mesocotyl tissue at the site of inoculation. On the basis of the results of the pathogenicity evaluation, MS-168 can be characterized as weakly virulent on seedling corn when grown under controlled environmental conditions. The identification of R. solani isolate MS-168 (AG-13) from corn in Mississippi broadens the natural distribution of occurrence and host range of this anastomosis group.

First Report of Stem Rot of Guiana Chestnut (Pachira aquatica) Caused by Pythium splendens.

Guiana chestnut is a perennial tropical plant that has recently become popular as a potted ornamental in Japan. In October 2001, severe stem rot occurred on Guiana chestnut plants grown in a greenhouse in Mie Prefecture, Japan. Water-soaked lesions appeared initially at the base of the stems and enlarged gradually toward the tops of plants. The affected tissues were softened and turned dark brown. Rotting was observed in the vascular bundles with advanced disease development. Globose hyphal swellings were numerous on diseased stems. Sections from diseased stems were cleaned by washing with running tap-water, placed on water agar, and incubated at 25°C. A species of Pythium was identified on the basis of morphological and cultural characteristics (1) and isolated consistently from the rotted stems of diseased plants. All isolates produced abundant hyphal swellings that were globose, smooth, 12 to 39 μm in diameter, mostly terminal, dark colored, and with dense granulated contents. Zoospores were absent. All isolates were of the compatibility '+ type' with production of sexual organs when paired with cultures of the '- type' tester isolate of Pythium splendens Braun (CBS462.48). Oogonia produced by crossings between Guiana chestnut isolates and isolate CBS462.48 were terminal or intercalary, globose, smooth-walled, and 32 to 38 μm in diameter. Antheridia were terminal, one to three per oogonium, sac-like, and diclinous. Oospores were single, aplerotic, globose, and 28 to 32 μm in diameter. The thickness of the oospore wall ranged from 1 to 2 μm. The internal transcribed spacer rDNA sequences of representative isolate OPU591 from Guiana chestnut matched those of CBS462.48 (similarity 99.2%) and have been deposited in GenBank under the Accession No. AY375242. Pathogenicity tests were conducted on potted Guiana chestnut plants (30 cm high and 7 to 10 cm in diameter at base of the stem) using isolate OPU591. A mycelial suspension from one culture, grown at 25°C for 7 days on water agar, was inoculated onto a single plant. Prior to inoculation, a wound (10 mm deep and 30 mm long) was made on the surface at the stem base on five plants. The mycelial suspension was poured onto the base of the stems of five wounded and five nonwounded plants. In addition, five wounded and five nonwounded, noninoculated plants were used as controls. Plants were maintained in a greenhouse for 8 weeks after inoculation. The temperature and relative humidity in the greenhouse ranged from 25 to 30°C and 65 to 75%, respectively. Dark-brown rotting developed on the stems of all wounded, inoculated plants by 20 days after inoculation. P. splendens was isolated from diseased tissues and found to be morphologically identical to the original isolate. This confirmed P. splendens as the causal agent of the disease. Disease did not develop on nonwounded inoculated plants or noninoculated plants. To our knowledge, this is the first report of P. splendens on Guiana chestnut. Potted plants of Guiana chestnut are often injured by frequent transplanting and transferring. Such injuries may have predisposed the plant to infection by P. splendens. Reference: (1) A. J. van der Plaats-Niterink. Page 1 in: Monograph of the Genus Pythium. Studies in Mycology Vol. 21, Centraalbureau Voor Schimmelcultures, Baarn, the Netherlands, 1981.

First Report of A1 Mating Type of Phytophthora ramorum in North America.

Phytophthora ramorum is known in Europe and the western United States (1). In Europe, it is found in nurseries and landscape plantings. In the United States, it has been confined to coastal forests, and in California, it is found in a few horticultural nurseries. All European isolates tested have been A1 mating type, while all North American isolates were A2 mating type (2). Amplified fragment length polymorphism markers also indicated that the populations on the two continents are distinct, and nearly all North American isolates are from one clone (Kelly Ivors, unpublished). In June 2003, P. ramorum was isolated from diseased Viburnum and Pieris spp. cultivars from a Clackamas County nursery in northern Oregon and diseased Camellia sp. cultivar from a Jackson County nursery in southern Oregon. Representative isolates were submitted to the American Type Culture Collection, Manassas, VA. As part of the effort to determine the origin of these new infestations, we tested the nursery isolates for mating type. Seven Oregon nursery isolates, three Oregon forest isolates (from the predominant North American clone), and two European isolates were paired. Agar plugs from 3-day-old colonies were placed in close proximity on carrot agar plates, and then the plates were examined for oogonia after 3 and 10 days as advised by C. M. Brasier (personal communication). Oogonia and antheridia typical of P. ramorum (2) formed when isolates from the Clackamas County nursery were paired with the Oregon forest isolates and also when isolates from the Jackson County nursery were paired with the European isolates. Gametangia also formed in pairings between Oregon forest isolates and European isolates, but not in any other combinations. We developed polymerase chain reaction (PCR) primers for four microsatellite loci and determined allele sizes for the same set of isolates (unpublished). Microsatellite alleles of the Clackamas County isolates were identical to the European tester isolates, and alleles of the Jackson County isolates were identical to the Oregon forest isolates. These results indicate that the recent Oregon nursery infestations are of separate origins. The Clackamas County isolates are A1 mating type and have microsatellite alleles like the European testers, but according to shipping records, the nursery has received no host nursery stock directly from Europe. However, host nursery stock has been received from a Canadian nursery. The Jackson County isolates are of A2 mating type and have microsatellite alleles like the forest isolates of Oregon, which is consistent with the reported origin of these plants from a California nursery. These preliminary microsatellite results need to be validated against a larger isolate set but are congruent with the mating type results. The Oregon nursery infestations highlight the dangers of unregulated or underregulated transport of host nursery stock from infested areas to noninfested areas. All host plants from infested nursery blocks at the affected Oregon nurseries have been destroyed by incineration, and a monitoring program has been implemented. Other host nursery stock on site has been taken "off-sale" pending verification that it is disease free, per the United States Department of Agriculture, APHIS requirements.

Characterization of diversity in Colletotrichum acutatum sensu lato by sequence analysis of two gene introns, mtDNA and intron RFLPs, and mating compatibility

A diverse collection of isolates identified as Colletotrichum acutatum, including a range of fruit-rot and foliar pathogens, was examined for mtDNA RFLPs and RFLPs and sequence variation of a 900-bp intron of the glutamine synthetase (GS) gene and a 200-bp intron of the glyceraldehyde-3-phosphate dehydrogenase (GPDH) gene. RFLPs of mtDNA, RFLPs of the 900-bp GS intron and sequence analysis of each intron identified the same seven distinct molecular groups, or clades, within C. acutatum sensu lato. Sequence analysis produced highly concordant tree topologies with definitive phylogenetic relationships within and between the clades. The clades might represent phylogenetically distinct species within C. acutatum sensu lato. Mating tests also were conducted to assess sexual compatibility with tester isolates known to outcross to form the teleomorph Glomerella acutata. Mating compatibility was identified within one clade, C, and between two phylogenetically distinct clades, C and J4. The C clade represented isolates from a wide range of hosts and geographic origins. J4 clade contained isolates from Australia or New Zealand recovered from fruit rot and pine seedlings with terminal crook disease. That isolates in two phylogenetically distinct clades were capable of mating suggests that genetic isolation occurred before reproductive isolation. No other isolates were sexually compatible with the mating testers, which also were in groups C and J4. Certain clades identified by mtDNA and intron analysis (D1, J3 and J6) appeared to represent relatively host-limited populations. Other clades (C1, F1 and J4) contained isolates from a wide range of hosts. Isolates described as C. acutatum f. sp. pineum were clearly polyphyletic.

First Report of a Blight Caused by Rhizoctonia solani on Anubias heterophylla in Italy.

Anubias heterophylla Engler, which is used in aquariums, is a species in the Araceae family native to western tropical Africa. A few nurseries in Italy produce plants for this market. During the spring of 2002, symptoms of blight were observed on Anubias plants grown in an ebb and flow soilless media system in a nursery in Sardinia, Italy. Approximately 10% of the plants were infected. Symptoms initially developed as water-soaked, zonate lesions on the base of stems. Symptoms progressed rapidly and affected the entire plant. Eventually the plant collapsed. The growing conditions (soilless cultivation, high plant density, temperatures at 22-24°C, and 95 to 98% humidity) were conducive to disease development. Isolations from infected leaf and stem tissue on potato dextrose agar (PDA) with streptomycin sulphate at 100 mg/l consistently yielded a fungus with mycelial and cultural characteristics of Rhizoctonia solani Kühn (2). The fungus was characterized as R. solani AG-IV based on hyphal anastomosis with several AG-IV tester isolates (1). Pathogenicity tests were conducted by placing 5-day-old mycelial plugs grown on PDA at the base of healthy A. heterophylla stems and maintaining the plants in a dew chamber at 18 to 22°C. After 7 days, symptoms of basal rot were observed on stems of inoculated plants, and the entire plants were wilted. Plants not inoculated remained healthy. Rhizoctonia solani AG-IV was reisolated from all inoculated plants. The pathogenicity test was repeated. To our knowledge, this is the first report of R. solani on A. heterophylla. Since there are only a few nurseries that grow Anubias, the economic impact of the disease in Italy is minimal.

First Report of Web Blight on Yellow-Sage (Lantana camara) Caused by Rhizoctonia solani in Europe.

Lantana camara is increasingly grown in northern Italy as a potted plant and contributes to the diversification of offerings in the ornamental market. During the spring of 2001, selections of L. camara cuttings growing at a commercial farm located at Albenga (Riviera coast) exhibited tan leaf spots of irregular size and shape. Spots were at first isolated, 4 to 8 mm in diameter, and later coalesced and affected the entire plant. Heavily infected leaves, stems, and branches became blighted and were killed. Infected rooted cuttings also eventually died. Diseased cuttings showed a progressive reduction (to less than 20%) in rooting ability. Isolations from infected leaves and stems on potato dextrose agar (PDA), supplemented with 100 mg/liter of streptomycin sulphate, consistently yielded a fungus with mycelial and cultural characteristics resembling Rhizoctonia solani. The fungal isolates were further characterized as R. solani Kühn AG-4 based on hyphal anastomoses with several AG-4 tester isolates. Pathogenicity tests were performed by placing 5-day-old-fungal mycelial plugs, grown on PDA, at the base of five healthy yellow-sage stems and holding plants in a dew chamber at 18 to 22°C. After 2 days, foliage blight appeared on leaves of inoculated plants, and after 3 days, stems also became infected and entire plants wilted. Five noninoculated plants remained healthy. The fungal pathogen was reisolated from all inoculated plants. R. solani has been observed on L. camara in the United States (1) and the Philippines (2). To our knowledge, this is the first report of R. solani on L. camara in Europe.

Conidium morphology of Curvularia geniculata and allied species

The species delimitation of some members of Curvularia with 4-septate conidia was studied. Conidial shapes and sizes were variable to some extent depending on isolates and herbarium materials examined. By crossing experiments, C. affinis, C. fallax, and C. senegalensis showed no reproductive isolation from C. geniculata. Some isolates obtained from culture collections and from fields were not fertile with tester isolates of C. geniculata. Their conidial morphology were somewhat different from C. geniculata. SEM observations also revealed their morphological difference from C. geniculata. Restriction fragment length polymorphism (RFLP) of total DNA was analyzed with several isolates of species belonging to the “geniculata” group of genus Curvularia. DNA fingerprinting with probes prepared from GPD-1 and tublin coding regions gave clear differences among them, resulting in different banding positions. The results of analyses also confirmed that C. senegalensis, C. affinis, and C. fallax are synonymous to Curvularia geniculata. Two populations having 4-septate conidia with warping hilum and one population with rough surface conidia were clearly different from C. geniculata.