V8 Juice Agar Medium Research Articles

Morphological Characterization of Corynespora cassiicola Isolates in Culture Media

Corynespora cassiicola threatens soybean and cotton production in Brazil. The objective of this study was to evaluate cultural and morphological aspects of C. cassiicola isolated from soybean and cotton of different Brazilian regions, in culture media. The isolates were grown in PDA (Potato Dextrose Agar) and V8 juice agar media. The characteristics evaluated were: color, aspect, and growth rate of mycelia, as well as production and dimension of conidia, and number of septa per conidium. Culture media and isolates were compared using the Kruskal-Wallis or Tukey’s test at 5% significance level. The mycelia of the isolates were predominantly dark gray and light brown. C. cassiicola isolates grew better in V8 juice agar medium, presenting a higher mycelial growth rate. In PDA medium, the production of conidia was higher in isolates from cotton, compared with soybean isolates. There was great variation in the production of conidia in V8 juice agar medium, regardless of the host origin. Conidia length and width varied for isolate and culture medium. The isolates of C. cassiicola coming from cotton presented a higher number of septa per conidium when grown in PDA medium. The morphological aspects of C. cassiicola vary depending on the host of origin and the culture medium.

Control of grey mould disease on strawberry using the effective agent, Bacillus amyloliquefaciens Y1

ABSTRACT This study was conducted to investigate the effect of Bacillus amyloliquefaciens Y1 on the control of grey mould disease caused by Botrytis cinerea and subsequent growth promotion of strawberry. Strain Y1 strongly inhibited mycelial growth of B. cinerea on different media with the highest inhibition of 61.22% on V8 juice agar medium. The higher the bacterial culture filtrate (BCF) concentration of strain Y1 blended in the PDA medium, the stronger the rate of inhibition on mycelial growth of B. cinerea. Microscopic observation revealed that the hyphal structure of the fungal pathogen was severely affected by the BCF indicating abnormal growth with excessive branching and swelling. Strain Y1 showed the promising ability to suppress grey mould disease under in vivo conditions and significantly lessened disease severity on flowers and fruits leading to an increase in disease control value when compared to fungicide treatment. Moreover, application of strain Y1 resulted in a significant promotion of strawberry plants with higher values in most growth characteristics compared to other treatments. The detection of higher levels of the defence-related enzyme, β-1,3-glucanase in the Y1-treated plants indicated that induction of resistance by this strain was at least partly involved in disease suppression. Even though there were reductions in the overall numbers of the soil bacterial population in the Y1-treated plants, no significant impact on the growth of strawberry was observed. Based on our results, strain Y1 is a promising candidate for environment-friendly disease control strategies.

Pathological Survey on Early Leaf Blight of Tomato and In Vitro Effect of Culture Media, Temperature and pH on Growth and Sporulation of Alternaria solani

Alternaria solani causing early blight of tomato is a serious disease, causing 50% to 86% losses in fruit yield and 20% to 40% losses in seedling establishment. Pathological survey, etiology and physiological characterization of the causal organism are necessary to formulate effective management strategy. Therefore, present study was conducted to determine the incidence and severity of early leaf blight of tomato in some selected area of Bangladesh and to evaluate the effect of different culture media on radial mycelial growth and sporulation as well as the effect of pH and temperature on mycelial growth. Survey was conducted in five upazilas of Bogura district and typical early blight disease sample was collected from surveyed field and Germplasm Centre, Agrotechnology Discipline, Khulna University, Khulna. Seven different culture media were used namely Potato Dextrose Agar (PDA), Richard’s Agar (RA), Waskman Agar (WA), Corn meal Agar (CA), Asthana and Hawker’s Agar (AHA), Oat Meal Agar (OMA), V-8 Juice Agar Medium (VJAM) and seven different pH level such as 5.0, 5.5, 6.0, 6.5, 7.0, 7.5 and 8.0 were used and also exercised five distinct temperatures such as 20°C, 25°C, 30°C, 35°C and 40°C to appraise cultural characters, mycelial growth and sporulation of A. solani. Cultural characters such as colony color, colony diameter, growth margin, growth surface and margin topography showed distinguished variation in culture media. Among the seven different media V-8 Juice Agar Medium gave not only maximum mycelial growth (73.10 mm) but also highest sporulation (2000 spores ml?1). The optimum pH for maximum mycelial growth ranged to 5.5 - 6.5. But maximum mycelial growth (71.70 mm) was observed in pH 6. Whereas, the mycelial growth was favored by 20°C - 30°C temperature but 25°C temperature provided highest mycelial growth (72.20 mm). However, temperature above 40°C was found adverse for the growth of tested fungus.

Morphological, cultural and physiological characters of Fusarium solani causing wilt of cluster bean

Cluster bean [Cyamopsis tetragonoloba (L.) Taub.], commonly called guar is an important legume crop belongs to family Fabaceae. It is cultivated mainly in the rainy season and major producing states in India are Rajasthan, Haryana, Gujarat, Maharashtra and to a limited extent in Uttar Pradesh and Madhya Pradesh. Cluster bean is a highly commercial, industrial and medicinally important leguminous crop due to presence of glactomannan gum in endosperm of seeds. The crop is known to suffer from many diseases which are responsible for its inferior quality and poor yield resulting in severe economic losses to the country as it is an important cash crop with a great potential for foreign exchange. In recent years Fusarium wilt of cluster bean has assumed a serious problem and causing considerable yield losses. Although wilt diseases has assumed importance in Maharashtra, no systematic work has been carried out so for the disease may appear in an epidemic form in future and may threaten the cultivation of cluster bean. Present studies were undertaken with objectives viz., isolation, identification, symptomatology, pathogenicity test, morphological, cultural and physiological characteristics of wilt disease. In cultural studies, it was observed that the test pathogen Fusarium solani shows maximum radial growth on Richard’s media and good sporulation was recorded on Richard’s, PDA and V8 juice agar media. In physiological studies, it was observed that the maximum growth and sporulation of Fusarium solani ranged from 25 °C to 30 °C and grew well from pH levels 6.5 to 7.

Cultural, morphological and bio-chemical variability of different isolates of Colletotrichum truncatum causing anthracnose of greengram

A laboratory experiment was conducted to study the variability among the eight isolates of Colletotrichum truncatum of greengram collected from different locations on the basis of cultural, morphological and biochemical characteristics by using nine different culture media viz., Potato Dextrose Agar (PDA), Potato Carrot Agar (PCA), Oat Meal Agar (OMA), Corn Meal Agar (CMA), Carrot Agar (CA), Sabouraud’s Agar (SA), Czapek’s Dox Agar (CDA), Richard’s Agar (RA) and V8 Juice Agar. Colony colour varied in different media from white or white with light brown centres which later changed to black or dark to light brown with increase in the age of the fungal cultures. Mostly, the colonies had fluffy or cottony mycelial growth with slight variations and regular to irregular white margin. PDA, PCA, CA and RA produced maximum mycelial growth (90 mm) at 10 DAI (days after inoculation). Minimum growth was observed on SA (69.56 mm) and V8 juice agar media (55.42 mm) and their difference was statistically significant. Morphological variability among the isolates was studied by comparing their conidial length, breadth and length and breadth of setae and their differences were statistically significant. Biochemical variability among the isolates was based on α- and β-esterase and peroxidise profiling. Positive activity was observed for both α- and β-esterase. α-Esterase enzyme showed the highest enzyme activity in terms of maximum numbers of banding loci among the three isozymes tested. The findings of the present study clearly revealed that cultural, morphological and biochemical variability did exist among the different isolates of C. truncatum.

First report of tobacco root rot caused by Phytophthora nicotianae in Mexico

During 2014 and 2015, symptoms of root rot were observed in tobacco fields located in Veracruz, Mexico. Phytophthora colonies developing rosaceous pattern were consistently isolated on potato dextrose agar (PDA) and V8 juice agar media. Sporangia were papillate, noncaducous, measuring 40 to 49 x 33 to 37 μm, and with short pedicels. The sporangial shape was ovoid or ellipsoid. Chlamydospores were mostly solitary, intercalary, or terminal, measuring 29-33 μm in diameter. The oomycete was identified as Phytophthora nicotianae based on descriptions of Gallegly and Hong (2008). To confirm the identification, genomic DNA from a representative isolate was extracted, and the internal transcribed spacer (ITS) region was amplified by PCR and sequenced using the primers ITS5 and ITS4 (White et al., 1990). The resulting 896 bp sequence was deposited as GenBank Accession No. KR906059. BLASTn analysis showed a 97% similarity with those of P. nicotianae (GenBank Accession No. AB688370). Pathogenicity tests were performed twice on 20 healthy tobacco plants by immersion of roots in a zoospore suspension adjusted to 1 x 104 zoospores ml-1. Ten tobacco plants inoculated with sterile distilled water served as controls. Inoculated plants developed root rot symptoms after 10 days, whereas no symptoms were observed on the control plants. The oomycete present on inoculated plants was morphologically identical to that originally observed on diseased plants, thus fulfilling Koch’s postulates. To our knowledge, this is the first report of tobacco root rot caused by Phytophthora nicotianae in Mexico.

First Report of Black Leaf Mold of Tomato Caused by Pseudocercospora fuligena in Ohio.

Diseased tomato (Solanum lycopersicum L. cvs. Geronimo, Rebelski, and Big Dena) plants were received for diagnosis from a home gardener in Wayne County, Ohio, in August 2013 and from a 0.14-ha greenhouse in Brown County, Ohio, in September 2013. Approximately 10 and 60% of leaf area was diseased in the home garden and greenhouse, respectively. One or more lesions, each with an indistinct border, were observed on the leaves. Black fungal growth was observed on both sides of the leaf in association with the lesions. Microscopic examination revealed Cercospora-like conidia (2). Three symptomatic leaves from each location were surface-sterilized with 0.5% NaClO for 1 min and cultured on V8 juice agar medium at room temperature under continuous fluorescent lighting. One isolate was selected from each of Wayne Co. (SAM33-13) and Brown Co. (SAM34-13). The fungus produced small, dark-brown colonies within 2 weeks of plating. Mycelium was olive brown and septate, producing fascicles of conidiophores. Conidia were cylindrical, 2 to 14 septate, and 25.8 to 109.7 × 6.5 μm. Genomic DNA was extracted from colonies of isolate SAM33-13 grown on V8 juice agar medium using the Wizard SV Genomic DNA Purification System (Promega, Madison, WI). The internal transcribed spacer (ITS) region of rDNA was amplified by PCR using primer pair ITS1 and ITS4 (5), and the purified amplicon was sequenced (OARDC Molecular and Cellular Imaging Center, Wooster, OH). The ITS sequence was 99% identical to those of GenBank accessions of Pseudocercospora fuligena from Korea (JX290079) and Thailand (GU214675). The sequence was deposited in GenBank (KF931141). Based on morphology (4) and sequence analysis, the fungus was identified as P. fuligena (Roldan) Deighton (basionym Cercospora fuligena). To satisfy Koch's postulates, three 4-week-old tomato plants each of the cultivars L390 (AVRDC, Taiwan) and Mountain Spring (Siegers Seed Co., Holland, MI) were sprayed with a suspension of 1 × 103 conidia/ml of isolates SAM33-13 or SAM34-13 prepared from 3-week-old cultures growing on V8 juice agar medium. Three non-inoculated control plants were sprayed with sterilized water. Plants were maintained in a growth chamber at 25 to 30°C, 80% RH, and a 12 h/12 h day/night cycle. The first symptoms appeared 3 weeks after inoculation as light yellow foliar lesions. The lesions enlarged and turned black due to fungal growth, and the infected leaves dried. Disease severity was 70 and 10% of leaf area for cvs. L390 and Mountain Spring, respectively, for each isolate. Non-inoculated control plants were symptomless, and no fungus was re-isolated from the leaves. P. fuligena was isolated from symptomatic leaves of inoculated plants as described above, and the identity was confirmed based on morphology. In the United States, C. fuligena has not been reported infecting tomato since the first report in Florida in 1974 (1). To our knowledge, this is the first report of black leaf mold of tomato caused by P. fuligena in Ohio. Resistant cultivars, crop sanitation, and fungicides are recommended to manage the disease (3).

First Report of Cherry Stem Rot and Leaf Necrosis Disease Caused by Phytophthora nicotianae in Yantai, China.

Cherry (Cerasus avium (Linn.) Moench) is the third most economically important fruit in Yantai, Shandong Province, China. In August 2012, brown spots or necrosis on cherry seedling leaves, with an incidence of 8.2 to 34.3%, were observed in some fields of cherry seedlings in Yantai. Our survey indicated that the economic losses could reach up to 15.3% if disease conditions, such as a cool rainy summer season, were favorable. Conspicuous watery lesions on the stems turned to brown streaks; the leaves all wilted; and finally the plants collapsed. Diseased stem and leaf samples were surface-disinfected in 1% sodium hypochlorite for 1 min, rinsed three times in sterile water, which was absorbed with filter paper, and then transferred to 10% V8 juice agar medium containing 50 μg/ml ampicillin and 5 μg/ml carbendazim (1). The plates were incubated at 22°C in the dark for 5 days. The colonies consisted of white, loose, fluffy aerial mycelia. Eight isolates were obtained, and all were identified as Phytophthora nicotianae based on morphological characteristics and the sequence of the internal transcribed spacer (ITS) region of rDNA. The sporangia were ovoid/spherical, obturbinate with rounded bases and prominent papillae that were 37.5 to 62.5 × 30 to 50 μm (average 46.4 × 37.8 μm, n = 100) in size, with an average length-to-breadth ratio of 1.2. Chlamydospores were terminal, intercalary, and measured 19 to 42 μm (average 30.4 μm), which is typical of P. nicotianae (2). The genomic DNA of the eight isolates was extracted from mycelia. The ITS region of all eight isolates was amplified using primers ITS1 and ITS4, producing specific products that were directly sequenced. The sequence of a representative isolate P1401 (895 bp) was submitted to GenBank (Accession No. KJ754387). It was 100% similar to P. nicotianae strains NV-20T and TARI 22073 (KC768775 and GU111667). To confirm the pathogenicity, at least 10 cherry leaves and new stems were inoculated with mycelial plugs (5 × 5 mm) from each isolate. Necrosis of leaves and stems was observed 4 and 7 days after inoculation, respectively. No symptoms were observed on the control leaves and stems that were inoculated with blank agar plugs. P. nicotianae was re-isolated from the infected leaves, and the ITS sequence was analyzed to confirm its identity. Phytophthora species, such as P. cambivora, P. megasperma, and P. drechsleri, had been previously isolated from cherry (3), but to the best of our knowledge this is the first report of stem rot and leaf necrosis disease caused by P. nicotianae on cherry. Since the economic loss caused by this disease could reach 15% if an outbreak occurred in a rainy summer, control measures should be implemented.

Improved production of phleichrome from the phytopathogenic fungus Cladosporium phlei using synthetic inducers and photodynamic ROS production by phleichrome

Two different diketopiperazines, cyclo-(L-Pro-L-Leu) and cyclo-(L-Pro-L-Phe), which were isolated from the culture filtrate of Epichloe typhina and found to be inducers of phleichrome production, were chemically synthesized and evaluated for use in the improved production of phleichrome from wild-type and UV-mutagenized strains (M0035) of Cladosporium phlei. When supplemented with PDA and V8 juice agar media, both inducers showed significant increases in the production of phleichrome. Phleichrome production was increased in a dose-dependent manner up to a concentration of maximum yield for both inducers. No further significant induction was observed by supplementing inducers over the concentration of maximum yield. Among the two inducers, cyclo-(L-Pro-L-Phe) showed better inducing capability than cyclo-(L-Pro-L-Leu). The maximum yield was observed from the M0035 strain grown on V8 juice media supplemented with 150 μM cyclo-(L-Pro-L-Phe), which was estimated to be 232.6 mg of phleichrome per gram of mycelia and 10.2 mg of secreted phleichrome per 20 agar-plugs. Interestingly, growth inhibition was observed on V8 juice agar media with 100, 150, and 200 μM cyclo-(L-Pro-L-Phe) but not on PDA with the same amount of inducer, which suggests that the inhibitory effect might be through the overproduction of phleichrome rather than the toxic effect of the inducer itself. Superoxide production by purified phleichrome was dramatically stimulated upon illumination, thus demonstrating photodynamic production of superoxide in vitro by phleichrome.

First Report of Pythium Root and Stalk Rot of Forage Corn Caused by Pythium arrhenomanes in Japan.

Corn (Zea mays L.) is the most important forage crop in Japan. It was cultivated on 92,000 ha in 2011 and was mainly used as whole crop silage for cattle feed. In September 2009, a root and stalk rot disease was detected on corn plants cultivated in Tochigi, located in the central region of Japan. The symptoms of the disease included wilting of whole plants after the R5 (dent) stage (2) with drooping ears. Roots turned black and their number decreased. Further, the stalks became hollow and soft and harbored white hyphae. This tissue deterioration made machine harvest difficult. We obtained seven isolates of a Pythium-like organism by single hypha isolation from surface-sterilized pieces of diseased roots and stems on water agar and deposited one of the isolates at the NIAS genebank, Japan, under the accession no. MAFF511547. The isolate was grown in the dark on V8 juice agar medium for 10 days to produce oogonia. The oogonia were globose, light brown to yellow, smooth, 23.9 to 30.5 μm in size, and had 1 to 8 antheridia. Oospores were mostly plerotic, and oogonia walls were 1.3 to 2.7 μm thick. The morphology of the isolates was similar to that of Pythium arrhenomanes Drechsler and consistent with the species description (3). We analyzed the rDNA-ITS region sequences of the isolate as described by Kageyama et al. (1). The sequence (GenBank Accession No. AB903904) showed 99.1% (783/790 bp) similarity with that of P. arrhenomanes (AY598628). On the basis of morphological and rDNA sequence similarities, we identified the isolates obtained from corn as P. arrhenomanes. The pathogenicity of the isolate was confirmed by planting corn seedlings of the commercial Pioneer Brand hybrid 36B08 immediately after germination in five replicate pots containing soil mixed with 5% boiled barley grain by weight, incubated with or without the isolate for 7 days. After 10 days of incubation in a greenhouse at 20 to 25°C, only the inoculated plants exhibited symptoms of root and stalk rot. Since the inoculated organism was readily re-isolated from the diseased stems and roots, the pathogenicity of the isolate was confirmed. For field observation, the same hybrid of forage corn was sown in the fields in Nasushiobara, Tochigi, on 16 May 2011. The hybrid was sown in a row of 2 m, with 20 seeds planted at a distance of 10 cm with two replicates. For inoculum, the isolate was cultured on 5-cm-long wooden toothpicks, previously soaked in potato dextrose broth and placed on a V8 agar plate for 7 days at 25°C in the dark until covered by hyphae. The toothpicks were pierced into wounds made on the stems of corn plants, approximately 10 cm above the ground, using a thin iron needle. The wounds were about 2 mm in diameter and 2 cm deep. Field inoculation was conducted in late July at the R1 (silking) growth stage. Disease symptoms were observed in mid-September at R5, and only those plants that were inoculated with the toothpicks harboring the hyphae exhibited the typical stem rot symptoms. To our knowledge, this is the first report of root and stalk rot caused by P. arrhenomanes in forage corn in Japan.

An Outbreak of Leaf Spot Caused by Stemphylium solani on Eggplant in Malaysia

In 2011, a severe gray leaf spot was observed on eggplant (Solanum melongena) in major eggplant growing areas in Malaysia, including the Pahang, Johor, and Selangor states. Disease incidence was >70% in severely infected areas of about 150 ha of eggplant greenhouses and fields examined. Symptoms initially appeared as small (1 to 5 mm diameter), brownish-black specks with concentric circles on the lower leaves. The specks then coalesced and developed into greyish-brown, necrotic lesions, which also appeared on the upper leaves. Eventually, the leaves senesced and were shed. Tissue cut from the edges of leaf spots were surface-sterilized in 1% NaOCl for 2 min, rinsed in sterilized water, dried, and incubated on potato dextrose agar (PDA). Fungal colonies were greyish green to light brown, and produced a yellow pigment. Single, muriform, brown, oblong conidia formed at the terminal end of each conidiophore, were each 21.6 to 45.6 μm long and 11.5 to 21.6 μm wide, and contained 2 to 7 transverse and 1 to 4 longitudinal septa. The conidiophores were tan to light brown and ≤220 μm long. Based on these morphological criteria, 25 isolates of the fungus were identified as Stemphylium solani (1). To produce conidia in culture, 7-day-old single-conidial cultures were established on potato carrot agar (PCA) and V8 juice agar media under an 8-h/16-h light/dark photoperiod at 25°C (4). Further confirmation of the identification was obtained by molecular characterization in which fungal DNA was extracted and the internal transcribed spacer (ITS) region of ribosomal DNA amplified using primers ITS5 and ITS4 (2), followed by direct sequencing. A BLAST search in the NCBI database revealed that the sequence was 99% identical with published ITS sequences for two isolates of S. solani (Accession Nos. AF203451 and HQ840713). The amplified ITS region was deposited in GenBank (JQ736023). Pathogenicity testing of a representative isolate was performed on detached, 45-day-old eggplant leaves of the cv. 125066-X under laboratory conditions. Four fully expanded leaves (one wounded and two non-wounded leaflets/leaf) were placed on moist filter paper in petri dishes, and each leaflet inoculated with a 20-μl drop of a conidial suspension containing 1 × 105 conidia/ml in sterilized, distilled water (3). The leaves were wounded by applying pressure to leaf blades with the serrated edge of forceps. Four control leaves were inoculated similarly with sterilized, distilled water. Inoculated leaves were incubated in humid chambers at 25°C with 95% RH and a 12-h photoperiod. After 7 days, symptoms similar to those observed in the original fields developed on both wounded and non-wounded inoculated leaves, but not on control leaves, and S. solani was reisolated consistently from the symptoms using the same method as the original isolations. Control leaves remained asymptomatic and the fungus was not isolated from these leaves. The pathogenicity testing was repeated with similar results. To our knowledge, this is the first report of S. solani on eggplant in Malaysia.

Leaf Spot on Lettuce (Lactuca sativa) Caused by Stemphylium solani, a New Disease in Malaysia.

In June 2011, lettuce (Lactuca sativa) plants cultivated in major lettuce growing areas in Malaysia, including the Pahang and Johor states, had extensive leaf spots. In severe cases, disease incidence was recorded more than 80%. Symptoms on 50 observed plants initially were as water soaked spots (1 to 2 mm in diameter) on leaves, and then became circular spots spreading over much of the leaves. In this research, main lettuce growing areas infected by the pathogen in the mentioned states were investigated and the pathogen was isolated onto potato dextrose agar (PDA). Colonies observed were greyish green to light brown. Single conidia were formed at the terminal end of conidiophores that were 28.8 to 40.8 μm long and 11.0 to 19.2 μm wide, and 2 to 7 transverse and 1 to 4 longitudinal septa. To produce conidia, the fungus was grown on potato carrot agar (PCA) and V8 juice agar media under 8-h/16-h light/dark photoperiod. Fourteen isolates were identified Stemphylium solani based on morphological criteria described by Kim et al. (1). To confirm morphological characterization, DNA of the fungus was extracted from mycelium and PCR was done using universal primers ITS5 (5'-GGAAGTAAAAGTCGTAACAAGG-3') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3'), which amplified the internal transcribed spacer (ITS) region of rDNA (2). The sequencing result was subjected to BLAST analysis which was 99% identical to the other published sequences in the GenBank database (GenBank Accession Nos. AF203451 and HQ840713). The nucleotide sequence was deposited in GenBank under Accession No. JQ736022. Pathogenicity testing of representative isolate was done using 20 μl of conidial suspension with a concentration of 1 × 105/ml in droplets (three drops on each leaf) on four detached 45-day-old lettuce leaves cv. BBS012 (3). Fully expended leaves were placed on moist filter paper in petri dishes and were incubated in humid chambers at 25°C. The leaves inoculated with sterile water served as control. After 7 days, disease symptoms were observed, which were similar to those symptoms collected in infected fields and the fungus was reisolated and confirmed as S. solani based on morphological criteria (1) and molecular characterization (2). Control leaves remained healthy. Pathogenicity testing was completed twice. To our knowledge, this is the first report of S. solani on lettuce in Malaysia and it may become a serious problem because of its broad host range, variability in pathogenic isolates, and prolonged active phase of the disease cycle. Previous research has shown that S. solani is a causal agent of gray leaf spot on lettuce in China (4).

Phylogenetic Identification of Pathogenic Fungi from Apple in Batu City, Malang, Indonesia

Pathogenic fungi cause dying off of apple tree and giving negative impact to farmers. The objective of this study was to identify tree pathogenic fungi isolates from apple tree in the Batu City and to know phylogenetic relationship of the isolates based on the DNA sequences. The method in this study included subculture of three pathogenic fungi isolates (M1, M4, and MB1) using V8 Juice Agar medium for one to three days, continued with DNA isolation, amplification using ITS5/ITS4 primer, purification, sequencing, and sequence homology analysis of DNA amplicons of each isolates with reference isolates. The results showed similarity value of DNA sequence of the three isolates M1, M4, and MB1 of more than 99%. The three isolates M1, M4, and MB1 have a similarity value of DNA sequence with species of Pythium splendens of 99.84%, 99.67% and 99.83%, respectively. On the other hand similarity value between those isolates with Phytophthora was less than 76%. It was concluded that the three isolates are Py. splendens.

First Report of Root Rot of Chicory Caused by Phytophthora cryptogea in Chile.

Chicory (Cichorium intybus L. var sativum Bisch.), a relatively new high-value crop in Chile, was introduced for commercial production of inulin. Inulins are polysaccharides extracted from chicory tap roots that are used in processed foods because of their beneficial gastrointestinal properties. Approximately 3,000 ha of chicory are grown for local processing in the BioBio Region near Chillan in south central Chile. Recently, a severe rot of 1 to 3% of mature roots in the field and after harvest has been observed in most fields, which caused yield and quality losses. Typical symptoms include a brown discoloration and a soft, watery decay of the root. Tissue pieces from symptomatic roots were placed on water agar and clarified V8 juice agar medium amended with antibiotics (1) for isolation of the causal pathogen. A Phytopthora sp. had been consistently isolated from root lesions, and axenic cultures were obtained using single-hypha transfers. The species was provisionally identified as Phytopthora cryptogea (Pethybridge and Lafferty, 1919) on the basis of morphological and cultural characteristics (1). Mycelia grew between 5 and 30°C with optimal growth at 20 to 25°C and no growth at 35°C. All isolates produced hyphal swellings and nonpapillate, persistent, internally proliferating, and ovoid to obpyriform sporangia with mean dimensions of 45 × 31 μm in sterile soil extract. The isolates were of A1 mating type because they produced oospores only when paired with reference isolates of P. cinnamomi A2 on clarified V8 juice agar amended with thiamine, tryptophan, and β-sitosterol (1) after 20 days at 20°C in the dark. On the basis of morphological and sequence data from cytochrome c oxidase subunit 1 and 2, internal transcribed spacer 2, and β-tubulin (GenBank Accession Nos. JQ037796 to JQ037798, respectively), the pathogen was identified as P. cryptogea. Pathogenicity tests were conducted using three isolates of P. cryptogea by placing a 7-mm-diameter disk from a 1-week-old V8 agar culture on 10 wounded and nonwounded healthy chicory roots (2). Control roots were mock inoculated with agar plugs. The inoculated roots were incubated at 20°C in a moist chamber. Root rot symptoms, identical to those observed both in field and storage, developed after 4 to 6 days only on wounded sites inoculated with the pathogen, and P. cryptogea was reisolated from these inoculated plants. Mock-inoculated roots remained healthy. This experiment was completed twice and similar results were obtained. To our knowledge, this is the first report of Phytophthora root rot of chicory caused by P. cryptogea in Chile.

First Report of Collar and Root Rot Caused by Phytophthora tentaculata on Witloof Chicory (Cichorium intybus) in Italy.

Witloof chicory (Cichorium intybus L.) is an important crop in Italy where most of the crop is still produced in soil. In September 2009, chicory plants (cv. Pan di Zucchero) grown on a commercial farm in Tarquinia (central Italy) showed symptoms of a previously unknown disease. Symptoms, observed 20 days after transplanting, consisted of stunting, yellowing of leaves, and a crown and root rot. Affected plants turned brown, wilted, and eventually died. At the soil level, dark brown-to-black water-soaked lesions coalesced and often girdled the stem. All of the crown and root system was affected. At this location, the disease was severe and widespread, with 60% of observed plants being affected. A Phytophthora-like organism was consistently isolated on a medium selective for oomycetes (4) after disinfestation of lower stem and root pieces of C. intybus for 1 min in a solution containing 1% NaOCl. Tissue fragments of 1 mm2 were excised from the margins of the root and crown lesions. The pathogen genus was identified as Phytophthora based on morphological and physiological features. Sporangia were produced for identification by growing a pure culture for 15 days on modified V8 juice agar medium (Campbell V8 juice [200 ml], agar [15 g], CaCo3 [0.5 g], and sterile water [800 ml]) under alternating light and dark (12/12 h). Sporangia were pyriform to ovoid, papillate, and measured 33.3 to 59.2 × 18.9 to 30.2 μm (average 39.9 × 25.8 μm). Chlamydospores developed in 28-day-old cultures and measured 21.3 to 30.2 × 19.5 to 29.7 μm (average 24.4 × 23.6 μm). Oogonia were globose and measured 26 to 41 μm (average 32.5 μm). Eighty percent of antheridia were paragynous. Amphyginous antheridia (15 to 20%) were also observed. Oospores were scarcely produced and measured 24 to 32 μm in diameter. The internal transcribed spacer (ITS) region of rDNA of a single isolate was amplified using the primers ITS4/ITS6 and sequenced. BLAST analysis (1) of the 851-bp segment showed 100% homology with the sequence of Phytophthora tentaculata. The nucleotide sequence has been assigned GenBank Accession No. GU949536. Pathogenicity of this isolate was confirmed by inoculating C. intybus cv. Pan di Zucchero plants 20 days after transplant. The same isolate was grown for 15 days on a mixture of 70:30 wheat/hemp kernels and then 5 g/liter of the inoculum was mixed into a substrate containing a mixture of blond and black peat (15:85 vol/vol), pH 5.5. Five plants per 2-liter pot were transplanted and four replicates were carried out. Twenty noninoculated plants represented the control treatment. The trial was repeated. Plants were kept in two growth chambers at two temperatures (20 and 25°C). Symptoms similar to those observed in the field developed 7 days after inoculation. Twenty days later, 100 and 40% of the plants were dead at 25 and 20°C, respectively. Control plants remained symptomless. P. tentaculata was consistently reisolated from symptomatic plants. To our knowledge, this is the first report of P. tentaculata on C. intybus in the world ( http://nt.ars-grin.gov/fungaldatabases/index.cfm ). P. tentaculata was recently reported on lavender in Spain (2) and oregano in Italy (3). The economic importance of this disease is relatively low on most commercial farms.

Phytophthora chrysanthemi sp. nov., a new species causing root rot of chrysanthemum in Japan

A new species of Phytophthora was isolated from stem and root rot of chrysanthemum in the Gifu and Toyama prefectures of Japan. The species differs from other Phytophthora species morphologically, and is characterized by nonpapillate, noncaducous sporangia with internal proliferation, formation of both hyphal swellings and chlamydospores, homothallic nature, distinctive intercalary antheridia, and funnel-shaped oogonia. The new species can grow even at 35°C, with an optimum growth temperature of 30°C in V8 juice agar medium. In phylogenetic analyses based on five nuclear regions (LSU rDNA; genes for translation elongation factor 1α, β-tubulin, 60 S ribosomal protein L10, and heat shock protein 90), the isolates formed a monophyletic clade. Although the rDNA ITS region shows a high resolution and has proven particularly useful for the separation of Phytophthora species, it was difficult to align the sequences for phylogenetic analysis. Therefore, ITS region analysis using related species as defined by the multigene phylogeny was performed, and the topology of the resulting tree also revealed a monophyletic clade formed by the isolates of the species. The morphological characteristics and phylogenetic relationships indicate that the isolates represent a new species, Phytophthora chrysanthemi sp. nov. In pathogenicity tests, chrysanthemum plants inoculated with the isolates developed lesions on stems and roots within 3 days, and the symptoms resembled the ones originally observed. Finally, the pathogen’s identity was confirmed by re-isolation from lesions of infected plants.

SPECIES IDENTIFICATION AND VARIATION IN THE NORTH AMERICAN CRANBERRY FRUIT ROT COMPLEX

Many different species of pathogenic fungi cause cranberry fruit rot. The contribution of any given species can be quite variable depending on a host of cultural and environmental factors. Control of fruit rot can be problematic in the Northeast and in other growing regions since losses due to fruit rot can be episodic. One possible cause for inadequate control is that pathogenic fungi typically have differential sensitivity to fungicides. Thus, variation in the species or populations of the fungi present can affect the success and quality of control. Our primary objective in this project was to determine the variation within five species of cranberry fruit rot pathogens (Phyllosticta vaccinii, Coleophoma empetri, Colletotrichum acutatum, Colletotrichum gloeosporioides, and Physalospora vaccinii) across geographic regions [NJ, MA, WI and British Columbia (BC)] using morphologic and molecular characters. Our secondary objective was to reexamine the taxonomy of these pathogens at the molecular level, particularly those that are cranberry- or Vaccinium species-specific. Our results suggest that morphologically, within-species variation was low. We did however collect atypical non-pigmented C. acutatum from BC and Physalospora vaccinii isolates from all regions varied from dark gray to pure white on V8 juice agar medium. Molecular analysis of ribosomal ITS sequences showed a low level of variability for most pathogen species, with the exception of the morphologically distinct isolates. Phylogenetic analysis showed that the atypical, non-chromogenic C. acutatum formed a distinct, but closely related clade, to the normal pink-pigmented isolates. In contrast, the pure white isolates of Physalospora vaccinii possessed an ITS sequence distinct from those of the gray strains, suggesting they may represent a different species.

Fitness of Isolates of Phytophthora capsici Resistant to Mefenoxam from Squash and Pepper Fields in North Carolina.

Despite the wide adoption of mefenoxam (Ridomil Gold EC) for vegetables in North Carolina, the incidence of Phytophthora blight on pepper (Capsicum annuum) and squash (Cucurbita pepo) is high. Seventy-five isolates of Phytophthora capsici were collected in five pepper and one squash field in order to assess mefenoxam sensitivity. The relative fitness of resistant and sensitive isolates was contrasted in vitro by their respective rates of colony growth and their ability to produce sporangia in unamended V8 juice agar medium. In in vivo experiments, the aggressiveness of isolates on pepper was evaluated. The frequency of resistant isolates in North Carolina populations was 63%, considerably higher than resistance levels in areas where mefenoxam is not widely adopted. Resistant isolates grew on amended media at rates >80 to 90% and >100% of the nonamended control at 100 μg ml-1 and 5 μg ml-1, respectively. Sensitive isolates did not growth at 5 or 100 μg ml-1. All isolates from three fields, including two pepper and a squash field, were resistant to mefenoxam. Populations from other fields were composed of either mixes of sensitive and resistant isolates or only sensitive isolates. Response to mefenoxam remained stable during the course of in vitro and in planta experiments. Occurrence of a mefenoxam-resistant population of P. capsici on squash is reported here for the first time in North Carolina. When measured by rate of colony growth, sporulation in vitro, or aggressiveness in planta, fitness of resistant isolates was not reduced. Mefenoxam-resistant isolates from squash were as aggressive on pepper as sensitive or resistant pepper isolates. These results suggest that mefenoxam-resistant populations of P. capsici are as virulent and fit as sensitive populations.

Phytophthora infestans Mating Types on Tomato (Solanum lycopersicum) in Southern Spain.

During 2004, an unusual spread of Phytophthora infestans on tomato plants in greenhouses located in Almería and Granada provinces, southern Spain, was observed. Infected plants had water-soaked, brown spots on leaves and stems and necrotic areas with white mold on the surface of fruits. Three isolates were obtained by plating diseased tissue on V8 juice agar medium and maintained on rye agar at 18°C. These isolates were analyzed for the mating type. Crosses were carried out using V8 juice agar and rye agar. The two parental isolates US1 (A1) and US8 (A2) were both provided by W. E. Fry, Cornell University, Ithaca, NY. Two of the Spanish isolates were homothallic and the other isolate belonged to the uncommon mating type A1A2. To confirm the occurrence of the two mating types, 43 single-sporangium progeny were produced and analyzed from the A1A2 mating type. Thirty eight isolates were A1, two were A2, one was A1A2 mating type, and two were sterile. Assessment of five single-sporangium progeny from the homothallic type resulted in two A1, two homothallic, and one sterile isolate. A1A2 isolates produced oospores when crossed with either A1 or A2, but not when self-crossed. Previously, the A1A2 mating type has been found in Israel in the field and was obtained from oospores produced on tomato seeds (2,3). Since 2003, mating types of P. infestans isolates recovered from potato (60) and tomato (8) in southern Spain have been characterized. Seventy-five percent of the isolates recovered from potato were A1 and 25% were A2 mating types. Isolates recovered from tomato were 50% A1 and 50% A2 (1). To our knowledge, this is the first report of the occurrence of the A1A2 mating type and homothallic P. infestans isolates on tomato in Spain.

Leaf Blight of Fatsia japonica caused by Phytophthora cactorum

A leaf blight caused by a species of Phytophthora was found on fatsia plants (Fatsia japonica Decne et Planch.) growing in an apartment garden in Daegu, Korea in late April to May, 2003. The species of Phytophthora isolated from the diseased plants produced sporangia and sex organs on V8 juice agar medium. Sporangia were papillate, ovoid to subspherical, and caducous with a pedicel. The dimensions of the sporangia were <TEX>$31.2-46.8\times23.4-33.2{\mu}m$</TEX> in range, <TEX>$39.6\pm4.1\times28.3\pm2.8{\mu}m$</TEX> in <TEX>$average{\pm}standard$</TEX> deviation, I/b ratio approximately 1.40, with papillae about <TEX>$3.6{\mu}m$</TEX> high, and pedicels <TEX>$0.9­5.8{\mu}m$</TEX> long. Oogonia were spherical, <TEX>$25.0-32.5{\mu}m$</TEX> in range with an average of <TEX>$28.2\pm2.3{\mu}m$</TEX> in diameter. Antheridia were predominantly paragynous, globose to ovoid, <TEX>$8.8-13.8\times7.5-10.0{\mu}m$</TEX> with an average of <TEX>$10.9\pm1.2 \times9.2\pm1.1{\mu}m$</TEX>. Oospores in the oogonia were aplerotic or plerotic, and <TEX>$20.0-25.0{\mu}m$</TEX> in diameter with an average of <TEX>$23.5\pm1.5{\mu}m$</TEX>. Pathogenicity of the isolate was confirmed on detached leaves of fatsia. The disease was observed only in April and May of 2003 when the weather was unusually wet. It then diminished with increase of temperature in the year and did not appear again on the same plants in 2004. Thus, the fungus appeared to be a relatively weak pathogen of fatsia.