Development of PCR Primers to Identify Fusarium oxysporum f. sp. fragariae.

Fusarium oxysporum f. sp. fragariae is a fungal pathogen causing Fusarium wilt on strawberry. Polymerase chain reaction (PCR) primers that can discriminate F. oxysporum f. sp. fragariae from nonpathogenic F. oxysporum would greatly assist pathogen identification. In order to develop a molecular diagnostic tool for this pathogen, transposable elements in the pathogen were characterized and used for designing a specific set of PCR primers. Portions of the transposable elements Fot3, Han, Hop, Hornet1, and Skippy were detected in all 33 strains of F. oxysporum f. sp. fragariae tested by PCR, whereas Foxy was detected in 32 strains and Impala sequences were detected in 30 strains. Two types of sequences were detected for Hop, two types for Impala, and three types for Skippy. The genomic region between Han and Skippy was amplified by an inter-retrotransposon amplified polymorphism technique, and PCR primers (FofraF and FofraR) to specifically identify F. oxysporum f. sp. fragariae were designed from this region. The developed PCR primers discriminated F. oxysporum f. sp. fragariae strains from nonpathogenic F. oxysporum strains and five other formae speciales. Conidia of F. oxysporum f. sp. fragariae could be detected in brown lowland-type soil by PCR using the primers. After preculturing the soil sample on FoG2 medium, 1 × 102 conidia/g of soil could be detected; without preculturing, 1 × 103 conidia/g of soil were detected.

First Report of Crown and Root Rot Caused by Binucleate Rhizoctonia AG-A on Thryptomene saxicola in Italy.

Thryptomene saxicola (Hook.) Schauer is an evergreen shrub native to Western Australia and a member of the Myrtaceae. In Italy, this species was recently introduced as an ornamental plant from abroad. From July of 2008 to September 2009, a new crown and root rot of T. saxicola was observed on several stocks of approximately 20,000 1- to 3-year-old potted plants. Diseased plants were obtained from a commercial nursery in eastern Sicily, Italy. They were propagated from cuttings and grown under drip irrigation. More than 30% of the plants showed disease symptoms. Infected plants were characterized by a lack of vigor. Roots and crowns were partially or completely destroyed, and as a consequence, infected plants were chlorotic and often wilted. Early in the disease development, roots and crowns showed brown lesions. Successively, mature crown lesions turned dark brown. Longitudinal sections of crown tissues revealed a discoloration of the basal stem. Diseased tissues were surface disinfested for 1 min in 1% NaOCl, rinsed in sterile water, plated on potato dextrose agar (PDA) amended with 100 mg/liter of streptomycin sulfate, and then incubated at 25°C. A binucleate Rhizoctonia (BNR) species was consistently isolated from affected tissues of plants. Phytophthora isolates were not recovered from symptomatic tissues plated on BNPRAH (benomyl, nystatin, pentachloronitrobenzene, rifampicin, ampicillin, and hymexazol) selective medium. Fungal colonies were white with floccose, aerial hyphae. Hyphal cells were determined to be binucleate when stained with 1% safranin O and 3% KOH solution (1) and examined at ×400. Anastomosis groups were determined by pairing isolates with five different tester isolates of BNR AG-A on 2% water agar in petri plates (3). Anastomosis was observed with all tester isolates. The rDNA-ITS of one isolate of BNR (DISTEF-TS1) was sequenced (GenBank Accession No. AB514570) (2). The sequence from this isolate exhibited 99% homology with BNR AG-A (GenBank Accession No. AY738628). Pathogenicity tests were conducted on potted, healthy, 1-year-old plants of T. saxicola. Forty plants were inoculated by placing 1/cm2 plugs of PDA from 5-day-old mycelial cultures near the base of the stem. The same number of plants was treated with 1/cm2 PDA plugs as controls. Plants were kept at 25°C and 95% relative humidity on a 12-h fluorescent light/dark regimen. Root and crown rots, identical to those observed in the nursery, appeared 45 days after inoculation, and 80% of the inoculated plants died within 4 months. Control plants remained healthy. Binucleate Rhizoctonia was reisolated from symptomatic tissues, completing Koch's postulates. To our knowledge, this is the first report in the world of BNR causing disease on T. saxicola. References: (1) R. J. Bandoni. Mycologia 71:873, 1979. (2) M. Hyakumachi et al. Phytopathology 95:784, 2005. (3) C. C. Tu and J. W. Kimbrough. Mycologia 65:941, 1973.

Induction of systemic resistance in Arabidopsis thaliana in response to a culture filtrate from a plant growth-promoting fungus, Phoma sp. GS8-3.

The plant growth-promoting fungus (PGPF), Phoma sp. GS8-3, isolated from a zoysia grass rhizosphere, is capable of protecting cucumber plants against virulent pathogens. This fungus was investigated in terms of the underlying mechanisms and ability to elicit systemic resistance in Arabidopsis thaliana. Root treatment of Arabidopsis plants with a culture filtrate (CF) from Phoma sp. GS8-3 elicited systemic resistance against the bacterial speck pathogen Pseudomonas syringae pv. tomato DC3000 (Pst), with restricted disease development and inhibited pathogen proliferation. Pathway-specific mutant plants, such as jar1 (jasmonic acid insensitive) and ein2 (ethylene insensitive), and transgenic NahG plants (impaired in salicylate signalling) were protected after application of the CF, demonstrating that these pathways are dispensable (at least individually) in CF-mediated resistance. Similarly, NPR1 interference in npr1 mutants had no effect on CF-induced resistance. Gene expression studies revealed that CF treatment stimulated the systemic expression of both the SA-inducible PR-1 and JA/ET-inducible PDF1.2 genes. However, pathogenic challenge to CF-treated plants was associated with potentiated expression of the PR-1 gene and down-regulated expression of the PDF1.2 gene. The observed down-regulation of the PDF1.2 gene in CF-treated plants indicates that there may be cross-talk between SA- and JA/ET-dependent signalling pathways during the pathogenic infection process. In conclusion, our data suggest that CF of Phoma sp. GS8-3 induces resistance in Arabidopsis in a manner where SA and JA/ET may play a role in defence signalling.

First Report of Crown and Root Rot Caused by Binucleate Rhizoctonia AG-A on Dodonaea viscosa in Italy.

Florida hopbush (Dodonaea viscosa (L.) Jacq.) is an evergreen bush or small tree native to Australia and a member of the Sapindaceae. During September of 2008, a crown and root rot of D. viscosa was observed on 1-year-old potted plants in a commercial nursery in eastern Sicily, Italy. More than 15% of the plants showed disease symptoms. Infected plants were characterized by a lack of vigor. Roots and crowns were partially or completely destroyed, and as a consequence, infected plants were often wilted. Early in the disease development, roots and crowns showed brown lesions. Successively, mature crown lesions turned dark brown. Diseased tissues were surface disinfested for 1 min in 1% NaOCl, rinsed in sterile water, plated on potato dextrose agar (PDA) amended with 100 mg/liter of streptomycin sulfate, and then incubated at 25°C. A binucleate Rhizoctonia (BNR) species was consistently isolated from affected tissue of plants. Fungal colonies were white with floccose, aerial hyphae. Hyphal cells were determined to be binucleate when stained with 1% safranin O and 3% KOH solution (1) and examined at ×400. Anastomosis groups were determined by pairing isolates with five different tester isolates of BNR AG-A on 2% water agar in petri plates (3). Anastomosis was observed with all tester isolates. The rDNA-ITS of one isolate of BNR (DISTEF-DV2) was sequenced (GenBank Accession No. AB514569) (2). The sequence from this isolate exhibited 99% homology with BNR AG-A (GenBank Accession No. AY738628). Pathogenicity tests were conducted on potted, healthy, 8-month-old plants of D. viscosa. Twenty plants were inoculated by placing 1/cm2 plugs of PDA from 5-day-old mycelial cultures near the base of the stem. The same number of plants was treated with 1/cm2 PDA plugs as controls. Plants were kept at 25°C and 95% relative humidity on a 12-h fluorescent light/dark regimen. Root and crown rots, identical to those observed in the nursery, appeared 30 days after inoculation, and all the inoculated plants died within 2 months. Control plants remained healthy. Binucleate Rhizoctonia was reisolated from symptomatic tissues, completing Koch's postulates. To our knowledge, this is the first report in the world of BNR AG-A causing disease on Florida hopbush. References: (1) R. J. Bandoni. Mycologia 71:873, 1979. (2) M. Hyakumachi et al. Phytopathology 95:784, 2005. (3) C. C. Tu and J. W. Kimbrough. Mycologia 65:941, 1973.

Population Structure of the Fusarium graminearum Species Complex from a Single Japanese Wheat Field Sampled in Two Consecutive Years.

The Fusarium graminearum species complex (Fg complex) that consists of at least 11 phylogenetically distinct species contains important Fusarium head blight (FHB) pathogens of wheat and barley worldwide. We obtained members of the Fg complex by sampling from a 500-m2 experimental wheat field in Kumamoto Prefecture, Japan in two consecutive years and assessed them for species identity and trichothecene chemotype. Haplotype diversity was estimated by using 11 variable numbers of tandem repeat (VNTR) markers. In addition to these two samples (group 03W in 2003 and group 04W in 2004), pathogen populations from seed that was harvested in Fukuoka Prefecture and planted in the experimental field in 2002 (group 02WSC) and pathogen populations from seed that was harvested in Nagasaki Prefecture and planted in 2003 (group 03WSC) were analyzed for this study. Forty-six isolates were collected in each group. Most isolates from wheat heads were classified as F. asiaticum; only four isolates were classified as F. graminearum sensu stricto (s. str.). Out of a total of 183 Fg complex strains, 80 isolates (44%) were of the NIV type, while 103 isolates (56%), including all four F. graminearum s. str. isolates, were of the 3ADON type. No 15ADON type isolate was detected in this study. Trichothecene chemotype compositions of 03W and 04W were nearly identical. High gene diversity of F. asiaticum was observed in all groups. Based on the observed low level of fixation index (FST) and high level of effective number of migrants (Nm), distinctive population subdivision of F. asiaticum was not inferred among the four groups. These results suggest that FHB in the experimental wheat field in both 2003 and 2004 was caused by a genetically similar population, which prevails in Kumamoto, Fukuoka, and Nagasaki prefectures.

First Report of Crown Rot, Stem Rot, and Root Rot Caused by Binucleate Rhizoctonia AG-G on Viburnum tinus in Italy.

Laurustinus (Viburnum tinus L.), native to the Mediterranean Region, is an evergreen shrub belonging to the Caprifoliaceae that is commonly cultured as an ornamental shrub or small tree. During the summer and autumn of 2007 and 2008, a widespread yellowing, partial foliar necrosis, or death of the whole plant was observed on 3- to 4-year-old potted plants of V. tinus in a commercial nursery in eastern Sicily (Italy). More than 20% of the plants showed disease symptoms. Infected roots, crowns, and stems turned dark brown, leaves gradually became necrotic, and infected plants were often killed. Diseased tissues were disinfested for 1 min in 1% NaOCl, rinsed in sterile water, plated on potato dextrose agar (PDA) amended with streptomycin sulfate at 100 mg/liter, and then incubated at 25°C. A binucleate Rhizoctonia (BNR) species was consistently isolated from affected tissue of Laurustinus. Fungal colonies were initially white, then turned light brown or brown with age, and formed irregularly shaped, light brown sclerotia after 10 days. Hyphal cells were determined to be binucleate when stained with 1% safranin O and 3% KOH solution (1) and examined with a microscope at ×400. Anastomosis groups were determined by pairing isolates on 2% water agar in petri plates (4). Pairings were made with tester strains of binucleate Rhizoctonia AG-A through AG-S, except AG-J and AG-M. Anastomosis was observed only with tester isolates of AG-G. The rDNA-ITS of two isolates of BNR (DISTEF-Vt 31 and DISTEF-Vt 32) was sequenced (GenBank Accession Nos. AB478783 and AB478784, respectively) (3). The sequence from these two isolates exhibited 100% homology with BNR AG-G (GenBank Accession No. AY927334). Pathogenicity tests were conducted on potted, healthy, 6-month-old laurustinus. Twenty plants were inoculated by placing 1-cm2 plugs of PDA from 5-day-old mycelial cultures near the base of the stem. The same number of plants was treated with 1-cm2 PDA plugs as control. Plants were kept at 25°C and 95% relative humidity with a 12-h fluorescent light/dark regimen. Stem, crown, and root rot symptoms, identical to ones observed in nursery, appeared 20 days after inoculation, and all the inoculated plants showed symptoms within 1 month. Control plants remained healthy. Binucleate Rhizoctonia was reisolated from symptomatic tissues, completing Koch's postulates. R. solani was previously reported on Viburnum sp. in the United States (2). To our knowledge, this is the first report of binucleate Rhizoctonia causing disease on V. tinus. References: (1) R. J. Bandoni. Mycologia 71:873, 1979. (2) D. F. Farr et al. Page 1252 in: Fungi on Plants and Plant Products in the United States. The American Phytopathological Society. St. Paul, MN, 1989. (3) M. Hyakumachi et al. Phytopathology 95:784, 2005. (4) C. C. Tu and J. W. Kimbrough. Mycologia 65:941, 1973.

Molecular characterization of the Fusarium graminearum species complex in Japan.

Members of the Fusarium graminearum species complex are important cereal pathogens worldwide and belong to one of at least nine phylogenetically distinct species. We examined 298 strains of the F. graminearum species complex collected from wheat or barley in Japan to determine the species and trichothecene chemotype. Phylogenetic analyses and species-diagnostic polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLPs) revealed the presence and differential distribution of F. graminearum sensu stricto (s. str.) and F. asiaticum in Japan. F. graminearum s. str. is predominant in the north, especially in the Hokkaido area, while F. asiaticum is predominant in southern regions. In the Tohoku area, these species co-occurred. Trichothecene chemotyping of all strains by multiplex PCR revealed significantly different chemotype compositions of these species. All 50 strains of F. graminearum s. str. were of a 15- or 3-acetyl deoxynivalenol type, while 173 (70%) out of 246 strains of F. asiaticum were of a nivalenol type. The possibility of gene flow between the two species was investigated by use of 15 PCR-RFLP markers developed in this study. However, no obvious hybrids were detected from 98 strains examined, including strains collected from regions where both species co-occur.

Control of Fusarium Crown and Root Rot of Tomato with Hypovirulent Binucleate Rhizoctonia in Soil and Rock Wool Systems.

Hypovirulent binucleate Rhizoctonia (HBNR) isolates L2, W1, W7, and Rhv7 were studied as potential antagonists of Fusarium crown and root rot of tomato (FCRR) caused by Fusarium oxysporum f. sp. radicis-lycopersici, in either soil or hydroponic rock wool systems. Reduction of FCRR on tomato by HBNR isolates was different depending on the isolate, days after inoculation of pathogen, and experiments. In the greenhouse soil system, HBNR isolates significantly (P = 0.01) reduced vascular discoloration and discoloration of total roots systems by 90 to 100% and by 73 to 89%, respectively, in three experiments. Under field soil conditions, HBNR W1 provided significant (P = 0.05) reduction of vascular discoloration by 71%. In the rock wool system, all HBNR isolates except L2 in experiment 1 significantly reduced (P = 0.05) vascular discoloration by 18 to 100% in four experiments. Plants treated with all HBNR isolates had foliar symptoms reduced by 41 to 100% in four experiments under the rock wool system. Application of HBNR also resulted in increases of marketable and total yields of tomatoes as much as 70 and 73%, respectively, over the untreated plants. The number of colony forming units of F. oxysporum f. sp. radicis-lycopersici per gram fresh weight of roots and stems was significantly reduced (P = 0.05) in plants treated with HBNR in both soil and rock wool systems. HBNR was re-isolated at a high frequency from roots grown inside paper pots containing soil infested with HBNR, but rarely isolated from the roots grown in soil infested with only F. oxysporum f. sp. radicis-lycopersici outside the paper pots. HBNR was not re-isolated from the tomato stems. Stem extracts from HBNR-treated and pathogen-challenged plants in the rock wool system inhibited germination and production of budding cells of F. oxysporum f. sp. radicis-lycopersici.

Characterization of a New Subgroup of Rhizoctonia solani Anastomosis Group 1 (AG-1-ID), Causal Agent of a Necrotic Leaf Spot on Coffee.

ABSTRACT A new foliar disease on coffee leaves was observed in Mindanao, Philippines, in 1996. The symptoms appeared as large circular or irregularly shaped necrotic areas with small circular necrotic spots (1 mm or less in diameter) usually found around the periphery of the large necrotic areas. Rhizoctonia solani was consistently isolated from these diseased coffee leaves. Isolates obtained were multinucleate (3 to 12 nuclei per hyphal cell), had an optimum temperature for hyphal growth at 25 degrees C, prototrophic for thiamine, and anastomosed with tester isolates belonging to R. solani anastomosis group 1 (AG-1). Mature cultures on potato dextrose agar (PDA) were light to dark brown. Sclerotia, light brown to brown, were formed on the surface of PDA and covered the whole mature colony culture. Individual sclerotia often aggregated into large clumps (3 to 8 mm in diameter) and their color was brown to dark brown. In pathogenicity tests, isolates from coffee caused necrotic symptoms on coffee leaves, whereas isolates of AG-1-IA (not isolated from coffee), 1-IB, and 1-IC did not. The results of analyses of restriction fragment length polymorphism of ribosomal DNA internal transcribed spacer, random amplified polymorphism DNA, and fatty acid profiles showed that R. solani isolates from coffee are a population of AG-1 different from AG-1-IA, 1-IB, and 1-IC. These results suggest that R. solani isolates from coffee represent a new subgroup distinct from AG-1-IA, 1-IB, and 1-IC. A new subgroup ID (AG-1-ID) is proposed.

Characterization of Rhizoctonia spp. Causing Root and Stem Rot of Miniature Rose.

Root and stem rot of miniature rose (Rosa hybrida L.) was observed in commercial glasshouse-grown roses in Gifu prefecture, Japan, during the summer and fall of 1997 and 1998. One hundred and fifty-three isolates of Rhizoctonia spp. were obtained from infected roots and stems. Of the 153 isolates, 9 had binucleate and 144 had multinucleate vegetative hyphal cells. Binucleate Rhizoctonia failed to anastomose with tester isolates of anastomosis groups (AG)-A through -S (not including AG-J and AG-M). Of 144 isolates identified as R. solani, 83.3% were AG 2-2 IIIB and 16.7% were AG 4 HG-I. Five isolates from each group caused severe rot and mortality on cuttings during rooting. Pathogenicity of Rhizoctonia spp. varied on three different ages of miniature roses cv. Silk. Isolates of AG 4 HG-I caused root and stem rot and mortality on 15-, 25-, and 40-day-old plants, whereas isolates of AG-2-2 IIIB caused root and stem rot and mortality on 15- and 25-day-old plants, but light root rot on 40-day-old plants. Isolates of binucleate Rhizoctonia caused root and stem rot and mortality only on 15-day-old plants.

Surface Disinfestation of Resting Spores of Plasmodiophora brassicae Used to Infect Hairy Roots of Brassica spp.

ABSTRACT Resting spores of Plasmodiophora brassicae were surface-disinfested by treatment with 2% chloramine-T for 20 min and then with an antibiotic solution (1,000 ppm of colistin sulfate, 1,000 ppm of vancomycin hydrochloride, and 6,000 ppm of cefotaxime sodium) for 1 day. The disinfested resting spores were used to inoculate hairy roots of cabbage (Brassica oleracea var. capitata cv. Fuji Wase), Chinese cabbage (B. pekinensis cv. Musou Hakusai), turnip (B. rapa var. rapifera cv. Wase Okabu), and rape (B. napus line Dc 119). Differences among hosts in susceptibility to clubroot in hairy roots were evident. Chinese cabbage and turnip hairy roots supported the highest percentages of root hair infection (53.3 to 80%) and the greatest production of zoosporangial groups (8.5 to 32.5 per root). Moreover, gall formation was observed only on Chinese cabbage and turnip hairy roots. The morphology of zoo-sporangia, plasmodia, and resting spores in diseased hairy roots was found to be identical to that in infected intact plants by both light and scanning electron microscopy. Pathogenicity tests confirmed the infectivity of resting spores produced in hairy roots. Thus, the hairy root culture technique should prove useful as a dual culture system for P. brassicae.

Isolation of Pythium Species from Zoysia Grass and Their Effect on Severity of Large Patch Disease.

Pythium periplocum, P. rostratum, P. torulosum, and P. vanterpoolii were predominant Pythium species isolated from nine sites with a history of large patch disease of zoysia grass. Rhizoctonia solani AG2-2 LP and the Pythium species were isolated from 21 sod samples of zoysia grass exhibiting large patch symptoms in five golf courses. R. solani AG2-2 LP was obtained from all samples, while P. periplocum, P. rostratum, P. torulosum, and P. vanterpoolii were obtained from 14, 6, 11, and 8 samples, respectively. At least one of the four Pythium species was recovered from 19 samples. To verify pathogenicity of these four species of Pythium on zoysia grass, they were inoculated alone and together with R. solani AG2-2 LP on zoysia grass. When individual isolates were used to inoculate zoysia grass, R. solani AG2-2 LP, P. periplocum, and P. vanterpoolii were moderately aggressive, while P. torulosum and P. rostratum caused little or no disease. Symptoms produced by R. solani AG2-2 LP included orange discoloration of the sheath, and the sheath was easily pulled from the crown. P. periplocum and P. vanterpoolii induced only sheath chlorosis, and the sheath was not easily removed from the crown. In coinoculation tests, the combination of R. solani AG2-2 LP and P. torulosum intensified disease severity on zoysia grass and induced more rapid symptom development than did R. solani AG2-2 LP alone. The combination of R. solani AG2-2 LP and P. periplocum or P. vanterpoolii resulted in sheath necrosis and bare patches, similar to large patch symptoms observed on golf courses.

Characterization of the Hyphal Swelling Group of Pythium: DNA Polymorphisms and Cultural and Morphological Characteristics.

The hyphal swelling (HS) group of Pythium species and P. ultimum were studied for cultural and morphological characteristics, restriction fragment length polymorphisms of the amplified internal transcribed spacer (ITS) region in nuclear rDNA, and random amplified polymorphic DNA (RAPD) analysis of genome DNA. The shape of sporangia was spherical to subspherical or lemoniform and averaged 18.1-23.0 µm. All isolates could grow at 5 to 35°C, and the rate at the optimal temperature, 30°C, was 29-34 mm/24 h. The size of the ITS region amplified by polymerase chain reaction and the banding patterns after digestion with the restriction enzymes showed no variation between the HS group and P. ultimum. No difference in banding patterns was shown between the HS group and P. ultimum by RAPD analysis with each of three primers. Isolates examined were from Japan, and results should be confirmed from other regions.

Characterization and Survival of Rhizoctonia solani AG2-2 LP Associated with Large Patch Disease of Zoysia Grass.

Prevalence and sites of survival of Rhizoctonia solani AG2-2 LP were studied in zoysia grass for 6 years. AG2-2 LP isolates commonly were recovered over all seasons at sites with a history of large patch disease. In patch margins, AG2-2 LP isolates were recovered from crowns of zoysia grass regardless of whether the disease occurred, but were most frequently isolated from the sheath tissues during disease occurrence. In healthy sites approximately 30 cm from the patch, isolates were obtained before but not during disease occurrence. Once disease occurred, patch symptoms rapidly expanded to the edge of tissue colonized by the pathogen during autumn to early spring. To verify that the pathogen spread to healthy areas, the clonal relationship among isolates was examined using their anastomosis reaction. Isolates recovered from the patch and healthy area outside the patch were of the same clone, but isolates from different patches differed. Cultural characteristics and pathogenicity of the AG2-2 LP isolates were compared with R. solani AG2-2 IIIB and R. solani AG2-2 IV. The AG2-2 LP isolates showed an irregular cluster of mycelia (not sclerotia), an irregular zonation, dark brown main hyphae, and sparse aerial hyphae on potato dextrose agar after 4 weeks of incubation. Optimum temperature for growth was 23°C. Cultural characteristics of AG2-2 subgroups IIIB and IV differed from LP isolates. All isolates of AG2-2 LP caused moderate to high levels of disease on zoysia grass, but were nonpathogenic or caused little disease on bent grass and sugar beet. These results indicate that cultural characteristics and host range of AG2-2 LP are different than those of AG2-2 IIIB and AG2-2 IV.

Carbon Loss and Germinability, Viability, and Virulence of Chlamydospores of Fusarium solani f. sp. phaseoli After Exposure to Soil at Different pH Levels, Temperatures, and Matric Potentials.

ABSTRACT (14)C-labeled chlamydospores of Fusarium solani f. sp. phaseoli were exposed to soil at 5, 15, 25, or 30 degrees C at pH 5 or 8 and water potential of -1 kPa or to soil at 0, -1, or -10 kPa at 25 degrees C at pH 6.9. Total carbon loss was greatest at 25 or 30 degrees C at pH 8 and -1 kPa. (14)CO(2) from respiration of chlamydospores and from soil microbes utilizing chlamydospore exudates accounted for the largest share of total carbon loss under all conditions. (14)(CO)(2) from soil microbial metabolism of (14)CO(2) exudates of chlamydospores was greatest in soil at 15, 25, and 30 degrees C, pH 8, and at either -1 or -10 kPa. Chlamydospore germinability in the absence of a C source (nutrient independence), viability in potato-dextrose broth, and virulence to kidney bean declined rapidly after exposure to soil at high temperatures (25 and 30 degrees C), pH 8, and the higher matric potentials (0 to-1 kPa). By contrast, germinability remained high (>50%), as did virulence, in soil at 5 degrees C and -10 kPa even after 70 days of incubation. Carbon loss was inversely correlated with germinability, viability, and virulence after exposure to soil at different pH levels, temperatures, and matric potentials.

Detection of Pythium ultimum Using Polymerase Chain Reaction with Species-Specific Primers.

This study was conducted to sequence the rDNA internal transcribed spacer (ITS) region of Pythium ultimum and Pythium group HS, design species-specific primers for polymerase chain reaction (PCR), and detect P. ultimum from diseased seedlings using PCR. The sequence of the ITS region of P. ultimum was identical with that of Pythium group HS. The results support the reports that the HS group is an asexual strain of P. ultimum. Using PCR, the primer pair K1+K3, designed on portions of the sequence of the ITS region, amplified isolates of P. ultimum and the HS group but not isolates of 20 other Pythium species. DNA extracts from damped-off seedlings were not amplified, but a 10-fold dilution of the extracts with Tris-EDTA (TE) buffer diluted the inhibitors and allowed PCR amplification. The primer pair used detected P. ultimum from a single diseased seedling.

Isolation of a DNA plasmid in the fungus Rhizoctonia solani.

A DNA plasmid, designated pRS64, was detected in three isolates of anastomosis group 4 (AG-4) of Rhizoctonia solani Kühn by biophysical methods. The plasmid was a linear double-stranded DNA with a molecular weight of 1.68 +/- 0.06 X 10(6) or 2617 +/- 87 bp. Weakly pathogenic isolates of R. solani, 1668 RI-1, 1271 RI-64 and 1272 RI-1, which showed abnormally slow growth, contained the plasmids, but pathogenic isolates, 1668, 1271 and 1272, showing normal growth, contained no detectable plasmid DNA.