Water-soaked Spots Research Articles

First Report of Acidovorax avenae subsp. citrulli as the Causal Agent of Bacterial Leaf Blight of Betelvine in Taiwan.

In November 2008, betelvines (Piper betle L., Piperaceae) exhibiting leaf blight symptoms were observed in central Taiwan. Infections resulted in a 30 to 70% loss of leaf yield in the investigated betel leaf-producing facilities. Symptoms began with small, necrotic, water-soaked spots that progressed to circular to irregularly shaped brown lesions, 5 to 10 mm in diameter, with chlorotic halos on leaves; some lesions started from the edge of leaves and later fused to form dried, necrotic margins. Bacteria-like streaming fluid was visible from the edges of freshly cut lesions at the junctions of chlorotic and necrotic leaf tissues when observed with a light microscope at ×100. When the streaming fluid was streaked onto King's medium B (3), a slow-growing, gram-negative, nonfluorescent bacterium was identified from the whitish colonies that consistently developed on the medium. Five bacterial isolates from three lesions were characterized with fatty acid methyl ester analysis (Agilent Technologies, Santa Clara, CA) and Sherlock Microbial Identification System (Microbial IDentification Inc., Newark, DE), and for each isolate, the bacterium was confirmed as Acidovorax avenae subsp. citrulli with a similarity index >0.70. In addition, the Biolog system (Biolog, Hayward, CA) and 16S ribosomal RNA sequence identity comparison were performed to confirm that the five betelvine-isolated bacteria were A. avenae subsp. citrulli based on a similarity of 0.54 with Biolog and 99% sequence identity for 16S rRNA gene. Koch's postulates were fulfilled by infiltrating a bacterial suspension of 3 × 105 CFU/ml into 40 leaves of four greenhouse-grown, disease-free, mature betelvine plants. After inoculation, plants were kept in a humidified greenhouse at 28°C to favor symptom development and symptoms similar to those observed in the greenhouse were evident at 7 days post inoculation (dpi) on all bacterium-infiltrated leaves. Control leaves infiltrated with distilled water remained symptomless. Bacteria showing morphological and biochemical similarities (2) to the ones used for inoculation were isolated from all of the inoculated betelvine leaves. In addition, a bacterial suspension at 3 × 108 CFU/ml was sprayed at the amount of 5 ml per plant onto 6 to 10 plants each of 4-week-old disease-free seedlings of watermelon (Citrullus lanatus (Thunb.) Matsum & Nakai, cv. Empire No. 2), oriental sweet melon (Cucumis melo L. var. saccharinus Naudin, cv. Silver Beam), and waxgourd (Benincasa hispida (Thunb.) Cogn., cv. Cheerer) for bioassays, and the inoculated seedlings were enclosed in plastic bags for 36 h at 28°C. Water-soaked lesions were observed on leaves of watermelon and waxgourd at 2 dpi and on sweet melon at 4 dpi on all inoculated plants but not on distilled water-sprayed control plants, indicating that A. avenae subsp. citrulli strains from betelvine could also infect melon plants. A. avenae subsp. citrulli was previously identified as the causal agent of bacterial fruit blotch on melon and bitter gourd in Taiwan (1). To our knowledge, this is the first report that A. avenae subsp. citrulli can naturally infect betelvine, a noncucurbit crop, to elicit bacterial leaf blight disease.

First Report of Xanthomonas hortorum pv. carotae Causing Bacterial Leaf Blight of Carrot in Mauritius.

Bacterial blight of carrot (Daucus carota) is caused by Xanthomonas hortorum pv. carotae (4). The pathogen is seed transmitted and carrot seeds can be an important source of primary inoculum (2). A 2008-2009 outbreak of a disease resembling bacterial blight was observed in Mauritius in 10 ha of carrot crops, primarily in humid areas of the island, at an estimated incidence of 10%. Carrot leaves with angular, water-soaked leaf spots that turned necrotic were collected at Plaine Sophie, Mauritius in December 2008. Yellow, Xanthomonas-like colonies were isolated onto KC agar medium (3). MultiLocus sequence analysis (MLSA) with four genes (atpD, dnaK, efp, and gyrB) was performed as described previously (1) on five carrot strains together with two reference strains of X. hortorum pv. carotae (LMG 8643 and LMG 8644). The reference strains were identical. Of the five Mauritius strains, two (LG1-1 and LG1-4) were identical, and most closely related to, but distinct from, the reference strains (genetic distance of 0.02). The other three strains represented two sequence types identified as Xanthomonas sp. based on a phylogenetic tree derived from concatenated sequences, but were not related to any type strain. PCR assays with a 3S primer pair specific for X. hortorum pv. carotae (2) produced an amplicon of approximately 350 bp from isolates LG1-1, LG1-4, and each of the reference strains. A PCR assay with a 9B primer pair (2) yielded an amplicon of 0.9 kb for strains LG1-1, LG1-4, and LMG 8644, whereas LMG 8643 yielded an amplicon of approximately 2.0 kb (2). Foliage of 4-week-old plants (36 plants per strain) of the carrot cv. Senator F1 were spray inoculated with a suspension of each strain using an 18-h culture in sterile 0.01 M tris buffer (pH 7.2) with approximately 1 × 108 CFU/ml. Plants sprayed with tris buffer were used as a negative control treatment. Plants were incubated in a growth chamber at 26 ± 1°C at a relative humidity of 95 ± 5% and a photoperiod of 16 h. Water-soaked lesions that developed into necrotic areas were observed 12 to 15 days after inoculation of LG1-1, LG1-4, and the two reference strains. Bacteria were recovered from lesions onto KC medium (3) 3 weeks after inoculation with mean Xanthomonas populations of at least 1 × 107 CFU/lesion. Colonies with morphology typical of Xanthomonas were recovered and typed using atpD sequencing to fulfill Koch's postulates. Although Xanthomonas-like bacteria were isolated from symptomatic carrot leaves in Mauritius in 1989, the results of that study were not published. To our knowledge, this is the first report of molecular and pathological characterization of this pathogen in carrot crops in Mauritius.

Goss's Bacterial Blight and Wilt of Corn Caused by Clavibacter michiganensis subsp. nebraskensis Occurs in Minnesota

Goss's bacterial wilt and blight caused by Clavibacter michiganensis subsp. nebraskensis (Vidaver and Mandel) Davis et al. can be an economically significant disease of corn (Zea mays L.) (1). Corn hybrids with typical leaf and wilt symptoms of Goss's bacterial blight were observed in two western Minnesota fields in Chippewa and Stephens counties in August 2009. Disease incidence was estimated at 40% in one field and 90% in the other. Symptoms consisted of large, tan-to-gray, linear lesions with irregular margins parallel to the veins, with up to 50% of the leaf area symptomatic. Irregular, dark green-to-black, water-soaked spots occurred in the lesions and dried bacterial exudate was present on the lesions. Bacterial streaming from the cut edge of lesions was visible with light microscopy. Fungal structures were not observed in the lesions. Bacteria were isolated from infected leaves collected in both fields. Sections were cut from the margins of the lesions and placed in 0.02 mM phosphate buffer (PB). Bacterial suspensions were spread onto yeast glucose medium (YGM) (3) and incubated for 5 days at 22°C. All colonies were orange and similar in appearance to C. michiganensis subsp. nebraskensis reference strain CIC016 (= CN313.0). Single colonies were subcultured onto YGM and CNS media. Two gram-positive strains, CIC251 and CIC252, were orange, circular, and convex on CNS medium and used to demonstrate Koch's postulates on corn (2). Bacterial suspensions containing 2 × 108 CFU/ml were prepared in PB from 5-day-old cultures grown on YGM. For each of strains CIC251 and CIC252, six plants of the hybrid DKC51-45 were inoculated at the V3 growth stage by swabbing inoculum over the second and third youngest leaves with Carborundum. Three control plants were treated similarly with sterile PB. Plants were incubated in a greenhouse at 20 to 24°C. Linear, water-soaked lesions typical of Goss's wilt began to develop on all inoculated leaves 7 days after inoculation. No symptoms developed on control plants. Two leaf samples with lesions were collected per plant and bacteria isolated as described above. Colonies with characteristics of C. michiganensis subsp. nebraskensis were isolated from all lesions. Presumptive identification of strains CIC251 and CIC252 as C. michiganensis subsp. nebraskensis, as well as colonies isolated from inoculated plants, was validated by rDNA sequencing. Genomic DNA was extracted from 3-day-old colonies on YGM and the 16S region was amplified (~1,480 bp) by PCR assay using primers F27 and r1492 (4). Forward and reverse sequences were aligned and base calls confirmed using Sequencher 4.9. Consensus sequences for each strain were compared with the nucleotide database with BLAST to confirm a 99% match to C. michiganensis subsp. nebraskensis (NCBI GenBank AM410697.1 and U09763.1). This confirms, for the first time (to our knowledge), that Goss's bacterial leaf blight and wilt of corn occurs in Minnesota and could be a production and phytosanitary concern in that state.

First Report on Almond in Europe of Bacterial Spot Disease of Stone Fruits Caused by Xanthomonas arboricola pv. pruni.

Symptoms characteristic of bacterial spot disease of Prunus spp. (4) were observed on almond trees (Prunus dulcis (Mill.) Webb) in 14 localities of Comunidad Valenciana (eastern Spain) and Aragón (northeastern Spain) between 2006 and 2009. Symptoms were first noted in the spring and were observed until leaf fall. Initial infections began on leaves as small, angular, water-soaked spots, which mainly developed toward the tip and along the leaf margins. These water-soaking lesions were surrounded by chlorotic tissue, although chlorosis did not extend more than a few millimeters. Subsequently, the lesions turned light brown, necrotic, and sometimes the necrotic spots fell out. When the lesions coalesced, they produced large necrotic areas. Sometimes premature leaf drop of infected leaves was observed in severely affected trees. Infected fruits initially displayed sunken, corky lesions that oozed gum, which later became raised when the mesocarp dehydrated. Infected fruits either dropped prematurely or remained on trees after harvest. Cankers typical of bacterial spot disease of stone fruit trees were observed on branches and shoots. Isolations from diseased leaves and fruits yielded Xanthomonas-like colonies on YPGA medium (yeast extract, peptone, and glucose agar), which were subsequently purified and characterized. All strains were gram-negative rods, oxidase negative, and strictly aerobic and showed typical biochemical characteristics of the Xanthomonas genus (3). A collection of 70 strains were further identified by PCR with primers Y17CoF/Y17CoR (1) as Xanthomonas arboricola pv. pruni by comparison with reference strains ISPaVe B4 and ISPaVe B6 isolated from Prunus salicina in Italy. A selection of 46 strains were also analyzed by immunofluorescence (IF) and ELISA using commercial polyclonal antibodies from NEOGEN Europe Ltd. (Ayrshire, Scotland, UK) and SEDIAG S.A.S. (Longvic, France), respectively), although ELISA antibodies proved to be not specific for X. arboricola pv. pruni. Pathogenicity was confirmed by inoculation of 70 almond strains and the reference strains on leaves of potted almond trees and/or on detached leaves (2) with bacterial suspensions (107 CFU per ml). One leaf was inoculated at 8 to 10 sites per strain. Characteristic bacterial spot disease symptoms (4) appeared on all inoculated leaves after 1 week of incubation at 25°C and high humidity, but not on the negative controls infiltrated with sterile distilled water. The original pathogen was reisolated from lesions of inoculated leaves and confirmed by biochemical tests, IF and PCR. As observed in Spain, the disease produces serious damage on the most susceptible almond cultivars like Antoñeta, Guara, Marta, Mas Bovera, and Vayro and can be very harmful, with severity of infection depending upon the relative cultivar susceptibility and environmental conditions. Appropriate eradication measures were taken after the causal agent was confirmed as X. arboricola pv. pruni. This pathogen was previously reported on almond in Japan and New Zealand (4). To our knowledge, this is not only the first report on almond in Spain but also in Europe.

Stem Rot of Garlic (Allium sativum) Caused by Sclerotium rolfsii

Stem rot disease was found in garlic (Allium sativum L.) cultivated from 2008 to 2010 in the vegetable gardens of some farmers in Geumsan-myon, Jinju City, Gyeongnam province in Korea. The initial symptoms of the disease were typical water-soaked spots, which progressed to rotting, wilting, blighting, and eventually death. White mycelial mats had spread over the lesions near the soil line, and sclerotia had formed over the mycelial mats on the stem. The sclerotia were globoid in shape, 1~3 mm in size, and tan to brown in color. The optimum temperature for growth and sclerotia formation on potato dextrose agar (PDA) medium was 30°C. The diameter of the hyphae ranged from approximately 4 to 8 μm. Typical clamp connection structures were observed in the hyphae of the fungus, which was grown on PDA medium for 4 days. On the basis of the mycological characteristics and pathogenicity of the fungus on the host plants, the causal agent was identified as Sclerotium rolfsii Saccardo. This is the first report of stem rot disease in garlic caused by S. rolfsii in Korea.

OCCURRENCE OF BACTERIAL SOFT ROT OF PLEUROTUS OSTREATUS CAUSED BY BURKHOLDERIA GLADIOLI pv. AGARICICOLA IN KOREA

An unknown bacterium was isolated from cultivated oyster mushrooms grown in Korea, which caused a soft rot that developed more rapidly than that elicited by the brown blotch bacterium, Pseudomonas tolaasii at temperatures above 25oC. From these lesions a bacterial strain (designated OM1) was isolated which, following inoculation of mushroom caps, yielded characteristic sunken water-soaked brown spots, which developed into a severe soft rot. In the early stages, the lesions were indistinguishable from those of bacterial brown blotch, well known to mushroom growers. Results of Gram stain and biochemical tests identified the new bacterial isolate as Burkholderia gladioli pv. agaricicola. This was confirmed by pathogenicity to oyster mushroom, physiological and biochemical properties, analysis of the 16S rRNA gene sequences and the fatty acids profile. This is the first report of the isolation of B. gladioli pv. agaricicola from cultivated oyster mushroom in Korea.

First Report of Bacterial Blight Caused by Pseudomonas viridiflava on Pea in Spain.

Because production of dry peas (Pisum sativum L.) is increasing in Spain, disease surveys were carried out from 2004 to 2006 in Castilla y Leon, the largest pea-producing region. In May of 2004, a leaf and stem blight caused an estimated 25% loss in yield in pea (cv. Messire) fields in El Cerrato (Palencia). Bacteria were isolated on King's B medium from 10 symptomatic plants from different fields (3). Thirty gram-negative isolates produced fluorescent, yellowish mucoid colonies. All isolates showed oxidative glucose metabolism on Hugh-Leifson medium and were levan and oxidase negative, potato soft rot positive, arginine dihydrolase negative, and tobacco hypersensitive positive. They also hydrolyzed esculine and gelatine. These results were different than those expected by Pseudomonas syringae pv. pisi and P. syringae pv. syringae (3). API 50 CH tests (bioMerieux, Marcy l'Etoile, France) revealed that all the isolates used the following carbon sources: glycerol, erythritol, l-arabinose, ribose, d-xylose, galactose, d-glucose, d-fructose, d-manose, inositol, manitol, sorbitol, d-raffinose, d-fucose, and d-arabitol. This nutritional profile is identical with that of P. viridiflava strain CFBP 6730, originally from pea plants in France. Therefore, these isolates were tentatively identified as P. viridiflava (2). Since a preliminary test demonstrated that 9 of the 30 isolates were pathogenic on pea plants, pathogenic isolates P44, P45, and P46 were selected arbitrarily for further tests. These three isolates plus strains HRI-W 1704 (P. syringae pv. pisi type race 6) and CFBP 1769 (P. syringae pv. syringae) were inoculated onto 10 pea seedlings (cv. Messire) each in two identical trials, following a described protocol (1). Seedlings inoculated with sterile distilled water were used as controls. After 10 days of incubation in a growth chamber at 22°C and 80% relative humidity, severe rotting and collapse similar to symptoms observed in fields appeared on pea seedlings inoculated with isolates P44, P45, and P46, while water-soaked leaf spots and necrotic symptoms were caused by P. syringae pv. pisi and P. pv. syringae. No symptoms were observed on plants inoculated with sterile water. Isolates recovered from symptomatic stems showed the same morphological and biochemical features of the original isolates. Sequences of 1,399 bp long from the three isolates (GenBank Accession Nos. GQ398128, GQ398129, and GQ398130) were 100% identical to P. viridiflava 16S rDNA database reference sequences. To our knowledge, this is the first report of P. viridiflava causing a disease of pea in Spain. The disease has been reported in New Zealand (4) and France (2).

Bacterial Spot of Tomato Caused by Xanthomonas perforans, a New Disease in Korea.

In July 2007, a leaf spot was observed on seedlings of tomato (Solanum lycopersicum) in a commercial greenhouse in Sungju County, Korea. Symptoms were dark, circular-to-irregular, water-soaked spots surrounded by chlorotic halos. Affected leaves turned yellow and readily detached. Two bacterial isolates, BC2642 and BC2923, were obtained from leaf lesions. The isolates were gram-negative, aerobic rods with a single flagellum. On peptone sucrose agar, colonies were yellow and raised with smooth margins. Starch and pectate hydrolysis tests were positive. Pathogenicity was confirmed by spraying cell suspensions containing 108 CFU/ml on seedlings of tomato (cv. Seokwang) and hot pepper (Capsicum annuum cv. Daekwang) in a greenhouse maintained at 28 ± 2°C. The isolates induced similar symptoms as those originally observed on tomato and also caused spots and a marginal blight of leaves of pepper 2 weeks after inoculation. No symptoms were noted on the control plants sprayed with sterilized distilled water. The identity of bacteria reisolated from spots on leaves of both plants were confirmed by comparison of patterns of metabolite fingerprints with those from preliminary identification of the isolates using the Biolog Microbial Identification System, version 4.2 (Biolog Inc., Hayward, CA), and reinoculation of the seedlings as above. The 16S rRNA gene (rrs) and the intergenic spacer (IGS) located between the rrs and the 23S rRNA gene, and partial sequences of gyrB were sequenced to aid in the identification of the isolates (1-3). A 2,134-bp fragment of the rrs and IGS regions and 701-bp fragment of the gyrB region from isolates BC2642 and BC2923 were compared with sequences in GenBank. Sequences from both isolates shared 100% similarity to sequences of Xanthomonas perforans (Genbank Accession No. AF123091). On the basis of the sequences and other assays, the two isolates were identified as X. perforans. To our knowledge, this is the first report of bacterial spot of tomato caused by X. perforans in Korea. Nucleotide sequence data reported are available under Accession Nos. GQ461739 and GQ461740 for rrs and IGS of BC2642 and BC2923, respectively, and GQ368187 and GQ380567 for gyrB of BC2642 and BC2923, respectively. An outbreak of this disease in the greenhouse may be due to the use of tomato seeds harvested in foreign countries where spot is known to occur. The disease is expected to have a significant economic impact on tomato culture in Korea.

Acidovorax avenae subsp. cattleyae에 의한 팔레놉시스 세균성갈색점무늬병의 발생

팔레놉시스 주요 재배지역인 경기, 부산에서 잎에 점무늬를 형성하며 병반의 가장자리는 선명한 노란색을 띄며, 잎 뒷면을 햇빛에 비춰볼 때 수침상을 나타내었고 병징이 심해지면 수침상 점무늬 병반이 합쳐지면서 잎 전체가 짙은 갈색으로 말라죽었다. 팔레놉시스 잎의 병반부로부터 세균을 분리하여 생리생화학적 방법, 병원성 시험결과 Acidovorax avenae subsp. cattleyae로 동정하였으며 이 병을 팔레놉시스 세균성갈색점무늬병으로 명명하였다. Leaf spot symptoms were observed in a commercial Palenopsis cultivated fields. Circular-elliptical watersoaked spots surrounded by a light green or yellow halo and turned to black when it was severe infection or blacken with gathered symptoms. These spots were finally enlarged to form of larger areas. Under the favorable conditions of temperature and moisture, the infection extended rapidly and dispersed to the crown and sometimes the infected plant became dead. A bacterial organism, isolated from the advancing margins of the lesions, was tested to characterize causing bacterium based on pathogenicity. The biochemical and physiological tests of that bacterium identified that as an Acidovorax avenae subsp. cattleyae. Therefore, we suggested to call that the new Phalaenopsis disease was bacterial brown spot caused by A.avenae subsp. cattleyae in Korea.

First Report of Xanthomonas cucurbitae Causing Bacterial Leaf Spot of Watermelon in the Seychelles.

Bacterial leaf spot of cucurbits caused by Xanthomonas cucurbitae (4) can be a harmful disease of several cucurbit species in tropical environments, mainly within the Cucumis, Cucurbita, and Citrullus genera. The bacterium induces angular, water-soaked leaf spots, which sometimes become necrotic and have a chlorotic halo. Scab-like lesions on fruit can also be observed (2). Water-soaked, angular leaf lesions were collected from approximately 15 watermelon plants (Citrullus lanatus) in a production field located in Mahé, Seychelles in 2003. Yellow-pigmented Xanthomonas-like bacterial colonies were isolated on KC semiselective medium (yeast extract 7 g, peptone 7 g, glucose 7 g, agar 18 g, distilled water 1,000 ml, propiconazole 20 μg ml-1, cephalexin 40 mg liter-1, and kasugamycin 20 mg liter-1) from all isolation attempts (3). Amplified fragment length polymorphism (AFLP) analysis was performed on four watermelon strains together with reference strains of Xanthomonas cucurbitae (LMG 690 [type strain] and LMG 8663) and the type strain of all other valid Xanthomonas species using SacI/MspI and four primer pairs (unlabeled MspI + 1 [A, C, T, or G] primers and 5'-labeled - SacI + C primer for the selective amplification step) (1). The four strains from watermelon showed identical fingerprints and were most closely related to X. cucurbitae. One strain from diseased watermelon (JZ88-1) was further analyzed by MultiLocus Sequence Analysis (MLSA) using portions of three housekeeping genes (atpD, dnaK, and gyrB) as described previously (1). This strain displayed a very high relatedness (99.8 and 98.9% with strain LMG 690 and LMG 8663, respectively) to the two reference strains of X. cucurbitae. AFLP and MLSA were useful for identifying strains at the species level that were consistent with previous results (1). Bottle-gourd (Lagenaria siceraria), pumpkin (Cucurbita maxima), squash cv. Aurore (Cucurbita pepo), cucumber cv. L-04 (Cucumis sativus), cantaloupe melon cv. Cezanne (Cucumis melo), and watermelon cv. Fou-nan (C. lanatus) leaves were infiltrated (10 inoculation sites per leaf and three replicates) with bacterial suspensions (JZ88-1, LMG 690 and LMG 8663) containing approximately 1 × 105 CFU ml-1 (approximately 1 × 102 CFU per inoculation site). Typical water-soaked lesions that developed into necrotic spots were observed 6 to 8 days after inoculation for all inoculated strains on all inoculated plant species. One month after inoculation, Xanthomonas was recovered from lesions and population sizes determined on KC semiselective medium (3) ranging from 1 × 106 to 9 × 106 CFU per lesion were typical of a compatible interaction. Bacterial leaf spot has appeared sporadically in Mahé, Seychelles since 2003, most often with limited incidence. However, growers need to be aware of the potential negative effect of this disease in tropical environments.

First Report of Bacterial Blight on Conventionally and Organically Grown Arugula in Nevada Caused by Pseudomonas syringae pv. alisalensis

In 2007, leaf spots were observed on arugula (Eruca vesicaria subsp. sativa cv. My Way) grown under sprinkler irrigation for fresh market in conventional and organic production fields located above 1,200 m (4,000 feet) in Nevada (NV). Approximately 30% of each planting was affected. Initially, symptoms consisted of small (<2 mm in diameter), angular, water-soaked spots visible from both sides of the leaf, some of which developed a shot-hole appearance. The spots enlarged and coalesced, remaining angular. Lesions ranged from black to tan, occasionally developing a chlorotic or purple margin. Some lesions resembled symptoms of downy mildew on arugula, but microscopic examination revealed no sporangiophores associated with the lesions. Bacterial ooze was observed when sections of symptomatic leaves were examined microscopically. Blue-green fluorescent pseudomonads were isolated from lesions on King's medium B agar from three arugula plantings. Twelve strains (at least three from each planting) were evaluated along with known strains of Pseudomonas syringae pv. alisalensis and P. syringae pv. maculicola in all assays. Bacterial strains were levan positive, oxidase negative, and arginine dihydrolase negative. Strains did not rot potato slices but induced a hypersensitive reaction on tobacco (Nicotiana tabacum L. cv. Sansun) indicating that the bacteria belonged to Lelliot's LOPAT group 1 (2). This was confirmed by analysis of fatty acid methyl esters (MIS-TSBA version 4.10; MIDI, Inc., Newark, DE), which indicated that the strains were highly similar (similarity >0.80) to P. syringae. Amplification of DNA between repetitive bacterial sequences (rep-PCR) using the BOXA1R primer resulted in identical banding patterns for the NV arugula strains and P. syringae pv. alisalensis from arugula in California (1). Koch's postulates were completed by confirming pathogenicity of the isolated strains on the arugula cvs. Italian and Astro. Strains were grown on nutrient agar for 48 h at 27°C, adjusted to 108 CFU/ml in sterile 0.01 M phosphate buffer (pH 7.0), and spray inoculated until runoff onto 2- to 3-week-old plants. Control plants were similarly sprayed with sterile phosphate buffer. Plants were held for 2 days in a mist chamber and 7 days on a greenhouse bench (24 to 26°C). Angular lesions similar to symptoms observed on the original plants developed on leaves of all inoculated arugula plants. In addition, some plants developed blackening of the smaller veins accompanied by chlorosis of the surrounding interveinal tissue in 10- to 20-mm diameter areas of the leaves. Small black lesions (as much as 10 mm long) were also observed on the petioles. Bacterial strains reisolated from the symptomatic tissue were identical to P. syringae pv. alisalensis by rep-PCR. Control plants remained symptomless. Similar inoculation and incubation methods confirmed that the host range of the NV arugula isolates was identical to that of known strains of P. syringae pv. alisalensis. The arugula and P. syringae pv. alisalensis isolates caused leaf spots on broccoli raab (Brassica rapa subsp. rapa cv. Sorento) and oats (Avena sativa cv. Montezuma). Pathogenicity tests were repeated. This confirms that the leaf spot observed on conventionally and organically grown arugula in NV was caused by P. syringae pv. alisalensis. To our knowledge, this is the first report of this disease on arugula in NV.

First Report of Xanthomonas cucurbitae Causing Bacterial Leaf Spot of Pumpkin on Réunion Island.

Bacterial leaf spot of cucurbits caused by Xanthomonas cucurbitae (3) can be of economic importance in tropical and subtropical production areas, most often within the Cucumis, Cucurbita, and Citrullus genera. The bacterium induces angular, water-soaked leaf spots, which sometimes turn necrotic with a chlorotic halo. Scab-like lesions on fruit may also be observed (1). During 2000, water-soaked, angular leaf lesions were collected from pumpkin (Cucurbita pepo) in a production field located at Petit Serré, Réunion Island. Yellow-pigmented Xanthomonas-like bacterial colonies were isolated on yeast peptone glucose agar. Amplified fragment length polymorphism analysis was performed on four pumpkin isolates together with reference strains of X. cucurbitae (LMG 690 [type strain] and LMG 8663) and the type strain of all other valid Xanthomonas species using SacI/MspI and four primer pairs (unlabeled MspI + 1 [A, C, T, or G] primers and 5'-labeled - SacI + C primer for the selective amplification step) (N. Ah-You, L. Gagnevin, P. A. D. Grimont, S. Brisse, X. Nesme, F. Chiroleu, L. Bui Thi Ngoc, E. Jouen, P. Lefeuvre, C. Verniére, and O. Pruvost, personal communication). The four isolates from pumpkin showed identical fingerprints and were most closely related to X. cucurbitae, with evolutionary genome divergences ≤0.05 (N. Ah-You et al., personal communication). One strain from diseased pumpkin (JW210-1) was further analyzed by multilocus sequence analysis using three housekeeping gene portions (atpD, dnaK, and gyrB) as described previously (N. Ah-You et al., personal communication). Although not fully identical, this strain displayed a similarity of >99% to the two reference strains of X. cucurbitae. Pumpkin and bottle-gourd (C. maxima), squash cv. aurore (C. pepo), cucumber cv. L-04 (Cucumis sativus), melon cv. cezanne (Cucumis melo), and watermelon cv. fou-nan (Citrullus lanatus) leaves were infiltrated (10 inoculation sites per leaf; three replicates) with bacterial suspensions prepared from strains JW210-1, LMG 690, and LMG 8663 and containing approximately 1 × 105 CFU ml-1. Negative controls consisted of leaves infiltrated with sterile tris buffer. Typical, water-soaked lesions that developed into necrotic spots were observed 6 to 8 days after inoculation for all inoculated plant species-strain combinations, but not for negative controls. One month after inoculation, mean Xanthomonas population sizes recovered from leaf lesions on KC semiselective medium (2) ranged from 1 × 107 to 1 × 108 CFU per lesion, typical of a compatible interaction. The reported outbreak was circumscribed to a single field and did not affect the local industry. No major outbreak of bacterial leaf spot of cucurbits has been reported on Réunion Island since 2000 on any host species of X. cucurbitae.

First Report of Anthracnose Fruit Rot Caused by Colletotrichum acutatum on Strawberry in Korea

Symptoms typical of anthracnose fruit rot; sunken, dark brown lesions on maturing fruits, were found in a commercial field of strawberry (Fragaria × ananassa) cv. Cal Giant in Yangyang County, Korea in May 2007. Masses of conidia were produced in acervuli in the center of lesions. The fungus was isolated on potato dextrose agar (PDA). Colonies grown on PDA were pale to mouse gray and became dark green to black in reverse. Conidia were formed in orange-to-salmon pink masses in the center of the culture. The average size of conidia on PDA was 15.2 × 4.6 μm, and they were hyaline, straight, cylindrical, with pointed ends, and aseptate (1). The fungus did not form an ascigerous stage in culture. Mycelial growth rate was 7.5 mm per day at 25°C on PDA. The identity of two isolates was confirmed as Colletotrichum acutatum J.H. Simmonds by PCR amplification using species-specific primers TBCA and TB5 (2), resulting in a characteristic 330-bp band on agarose gel. Morphological characters were in accordance with previous reports on C. acutatum. A pathogenicity test was conducted with five healthy plants of cvs. Cal Giant, Maehyang, Seolhyang, Kumhyang, Akihime, and Redpearl. After fruits and flowers were sprayed with a conidia suspension (105 conidia per ml), the plants were maintained at 10 to 25°C and 100% relative humidity in a greenhouse. As a control, five healthy plants were sprayed with sterile distilled water and incubated under the same conditions. Dark brown, water-soaked spots appeared on mature fruits of all cultivars after 5 days, and lesions on green fruits appeared on individual achenes. Flowers developed dark lesions, dried out, and died. No symptoms were found on the control plants. After the pathogen was reisolated from fruits and flowers lesions, the morphological characters developed in culture as described above. To our knowledge, this is the first report of C. acutatum causing strawberry anthracnose in Korea.

First Report of Leaf Spot Caused by Periconia igniaria on Yellow Starthistle in Russia.

Yellow starthistle (YST), Centaurea solstitialis L., is a weedy plant that is widely distributed in the Krasnodar Region of Russia. It is also an aggressive invasive weed in the western United States and a target of biological control efforts. In the summer of 2006, several hundred diseased plants were found near Taman, Russia. Symptoms of the disease were yellow, water-soaked leaf spots. Diseased leaves were collected, air dried, and sent to the Russian State Collection of Phytopathogenic Organisms at the All Russia Institute of Phytopathology (ARIP). The fungus isolated from the diseased leaves conformed to Periconia igniaria E.W. Mason & M.B. Ellis (teleomorph Didymosphaeria igniaria C. Booth) (1). Colonies of the fungus grew rapidly on potato glucose nutrition medium with aerial mycelium from fluffy to pressed and colorless at the beginning and darkening to black with age. The medium side of the colonies gradually became violet purple to wine colored. Conidiophores had aerial mycelia as much as 550 μm long and 9 to 13 μm wide tapering to 6 to 10 μm. Conidiophores were dark with short, swollen branched stipes. Conidia, formed in short twisted chains, were spherical, dark brown, 7 to 9 μm in diameter, and covered by 1 μm long spines. Yellow starthistle plants were grown in growth chambers with day/night air temperatures of 26 to 28/20 to 22°C, 60 to 70% relative air humidity, and 10,000 lx light for 16 h. Fifteen plants in the rosette stage were spray inoculated with an aqueous suspension of P. igniaria conidia at 5 × 106 conidia/ml and 5 ml per plant. Disease on leaves was observed on all plants 3 to 4 weeks after inoculation when the plants started to bolt. When the plants reached flowering stage, diffused yellow spots were observed on stems and inflorescences and all flowers died. Diseased leaves were surface disinfested and put on potato saccharose nutrition medium. P. igniaria was reisolated from 3 to 5 leaves of each plant and from flowers and stems that developed from 10 inoculated rosettes. Flowers of 10 YST plants were also inoculated with P. igniaria isolated from the previously inoculated plants. Disease developed in the flowers of all inoculated plants, and the symptoms were identical to those observed when rosettes were inoculated and disease followed bolting and flowering. No symptoms developed on four noninoculated plants included in each test. Internal transcribed spacer (ITS) sequences of the fungus were obtained and compared with sequences from GenBank. An uncultured soil fungus and three isolates of P. macrospinosa Lefebvre & Aar.G. Johnson produced the best homology (96%). No sequences for P. igniaria were available for comparison, but the description of P. macrospinosa (conidia 18 to 32 μm in diameter with 2.5 to 6 μm long spines) is clearly different than our isolate. ITS sequences for our isolate have been deposited in GenBank (Accession No. EU367468) and a voucher specimen has been deposited with the U.S. National Fungus Collection (BPI 878355). To our knowledge, this is the first report of P. igniaria causing disease on YST. Live cultures are being maintained at the Russian State Collection of Phytopathogenic Organisms in ARIP. Reference: (1) M. B. Ellis. Dematiaceous Hyphomycetes. CMI, Kew, UK, 1971.

First Report of Blackleg of Ajuga multiflora Caused by Phoma multirostrata in China.

Ajuga multiflora Bunge (Labiatae) is an ornamental that is widely planted in gardens in southern China. Blackleg symptoms were observed on A. multiflora during the autumn of 2005 in the Hangzhou area, Zhejiang Province. A. multiflora had to be replaced by other landscape plants during the past 2 years because of a serious outbreak of the disease. When the plant is in continuous cultivation for more than 2 years, disease incidence is more than 80%. Brown water-soaked spots appeared initially on the base of the petioles. Blackleg lesions from multiple infections may coalesce. Lesions often expand rapidly along the petioles followed by wilting and drying of individual leaves, ultimately resulting in the death of plants. The disease can occur at any time of year, but is most severe during the flowering stage. A fungus was consistently isolated from symptomatic tissues on potato dextrose agar. After 14 days on SNA (synthetic low nutrient agar; 1.0 g of KH2PO4, 1.0 g of KNO3, 0.5 g of MgSO4·7H2O, 0.5 g of KCl, 0.2 g of glucose, 0.2 g of sucrose, and 20 g of agar per liter) medium, the fungus produced light olive-to-gray mycelium, and later, generated brown, globose to subglobose pycnidia with hyaline, unicellular, and ellipsoidal conidia. Diameter of pycnidia ranged from 163 to 260 μm (average 203 μm). Conidiophores measured 3.5 to 6.2 × 2.0 to 3.2 μm (average 5.4 × 2.3 μm). The fungus was identified as Phoma multirostrata (P.N. Mathur, S.K. Menon & Thirum) Dorenb. & Boerema based on its morphological characteristics (1). The fungus grew between 4 and 36°C and optimum growth was at 23°C. To fulfill Koch's postulates, 15 healthy 20-day-old A. multiflora seedlings were planted in sterile soil artificially infested with P. multirostrata, which was grown on a mixture (1:10 w/w) of corn meal and sand. Soil was amended with 20% (w/w) of inoculum mixture. Another 15 plants grown in noninfested soil served as controls. All plants were grown at 22 to 25°C in the greenhouse. After 5 to 7 days, inoculated plants showed symptoms closely resembling those seen on plants from the garden. P. multirostrata was isolated from the margins of necrotic tissues on diseased plants, but not from the control plants. To our knowledge, this is the first report of P. multirostrata causing blackleg of A. multiflora in China. Reference: (1) B. C. Sutton. The Coelomycetes. CMI, Kew, Surrey, UK, 1980.

Identification of a highly virulent strain of Xanthomonas axonopodis pv. malvacearum

A highly virulent strain (HVS) of Xanthomonas axonopodis pv. malvacearum (Xam) was first reported in Africa in 1983, infecting all commercial cultivars of cotton including the immune cv. ‘101–102B’. The HVS was considered to be a new race of pathovar malvacearum (race 20). Here we studied a HVS (GSPB 2388) isolated in Sudan, which causes symptoms that clearly differed from the typical angular water-soaked spots of bacterial bright of cotton. Our investigations showed that extracellular cellulase activity of this HVS was higher than that of the control strain GSPB 1386 (race 18). Additionally, SDS-PAGE indicated that the HVS cell wall contained short LPS molecules with fewer O-chain repeating units, lacking in GSPB 1386. The higher cellulase activity and the distinct lipopolysaccharide of HVS are correlated with the higher virulence and deviating symptom formation. Rep-PCR fingerprinting showed that the HVS was very closely related to other strains of Xam.

First Report in Myanmar of Xanthomonas axonopodis pv. mangiferaeindicae Causing Mango Bacterial Canker on Mangifera indica.

Bacterial canker of mango (or bacterial black spot) caused by Xanthomonas axonopodis pv. mangiferaeindicae (1) is a disease of economic importance in tropical and subtropical producing areas. X. axonopodis pv. mangiferaeindicae can cause severe infection in a wide range of mango cultivars and induces raised, angular, black leaf lesions, sometimes with a chlorotic halo. Several months after infection, leaf lesions dry and turn light brown or ash gray. Severe leaf infection may result in abscission. Fruit symptoms appear as small, water-soaked spots on the lenticels. These spots later become star shaped, erumpent, and exude an infectious gum. Often, a "tear stain" infection pattern is observed on the fruit. Severe fruit infections will cause premature fruit drop. Twig cankers are potential sources of inoculum and weaken branch resistance to winds (2). Suspected leaf lesions of bacterial canker were collected from mango nursery stock cv. Yin Kwe at a nursery in Yangon, Myanmar during March 2007. Nonpigmented Xanthomonas-like bacterial colonies were isolated on KC and NCTM3 semiselective agar media (4). Amplified fragment length polymorphism analysis was performed on three isolates from Myanmar and additional reference isolates of xanthomonads originating from Anacardiaceae (X. axonopodis pv. anacardii, X. axonopodis pv. mangiferaeindicae, X. axonopodis pv. spondiae, and X. translucens strains from pistachio) using SacI/MspI and four primer pairs (unlabeled MspI + 1 [A, C, T, or G] primers and 5'-labeled - SacI + C primer for the selective amplification step) (1). On the basis of multidimensional scaling (1), the Myanmar isolates were identified as X. axonopodis pv. mangiferaeindicae and were most closely related to group B strains that were isolated from mango in India and Eastern Asia (2). Mango cv. Maison Rouge leaves, inoculated as previously reported (1) with the Myanmar isolates, showed typical symptoms of bacterial canker 1 week after inoculation. One month after inoculation, mean X. axonopodis pv. mangiferaeindicae population sizes ranging from 5 × 106 to 8 × 106 CFU per lesion were recovered from leaf lesions, typical of a compatible interaction (1). Mangifera indica L. probably evolved in the area that includes northwestern Myanmar (3) and to our knowledge, this is the first confirmed detection of X. axonopodis pv. mangiferaeindicae from Myanmar. Further surveys and strain collection will be necessary to evaluate its geographic distribution and prevalence in the country.

First Report of Sclerotinia sclerotiorum on Buckwheat (Fagopyrum esculentum) in Italy

In each of two fields of buckwheat (Fagopyrum esculentum L.) grown in Benevento Province (southern Italy), 60 to 70% of the plants developed severe root and crown rot. Symptoms included irregular, water-soaked spots on stems that were eventually covered with cottony mycelia as the lesions enlarged. Black sclerotia usually developed within the mycelium. The fungus was isolated on potato dextrose agar and 2% water agar. On the basis of colony morphology, including the production of black sclerotia (1), the fungus was identified as Sclerotinia sclerotiorum (Lib.) De Bary. The identity of the fungus was confirmed by near exact identity of internal transcribed spacer sequences (99%) with two isolates of S. sclerotiorum in GenBank (Accession Nos. Z73800 and Z73799). Pathogenicity of the fungus on buckwheat was evaluated by transplanting 20 20-day-old healthy plants in a mixture of soil and fungal inoculum (0.5% of wet millet seeds colonized by four isolates of S. sclerotiorum). Lesions on crowns and roots developed after 12 days and sclerotia appeared approximately 20 days later. No symptoms developed on noninoculated plants. Reisolation from inoculated plants yielded colonies of S. sclerotiorum. To our knowledge, this is the first report of S. sclerotiorum on buckwheat in Italy. The high incidence and severity of the disease may be limiting factors in the development of buckwheat as an alternative crop of tobacco in southern Italy. Reference: (1) J. E. M. Mordue and P. Holliday. Sclerotinia sclerotiorum. No. 513 in: Descriptions of Pathogenic Fungi and Bacteria. CMI, Kew, Surrey, UK, 1976.

BACTERIAL LEAF SPOT OF CHRIST’S THORN, A NEW DISEASE CAUSED BY ACIDOVORAX AVENAE SUBSP. CITRULLI IN IRAN

During spring and summer 2004 and 2005, a new disease of Christ’s thorn (Paliurus spina-christi P. mill.) was observed in Iran, in some areas of the Kurdistan province. Symptoms were initially yellow areas on the leaves, in which irregular water-soaked spots 3-5 mm in diameter developed. A non-fluorescent bacterium was consistently isolated from lesions on King’s medium B. Fifteen bacterial isolates were obtained from different locations of west Iran. According to phenotypic, biochemical and physiological properties all bacterial isolates were identified as Acidovorax avenae subsp. citrulli. They produced round white colonies 1-2 mm in diameter, were all aerobic, gram-, levan-, arginine dihydrolase-, catalase- and potato soft rot-negative, but oxidase- positive, and induced hypersensitivity reaction in tobacco leaves. Pathogenicity of selected isolates was confirmed by injecting bacterial suspension into the underside of leaves. This is the first report of a bacterial disease on Christ’s thorn in Iran.

Angular Leaf Spot of Cucumber Caused by Pseudomonas syringae pv. lachrymans in Kurdistan.

During the spring of 2004 and 2005, angular leaf spot was observed on cucumber (Cucumis sativus L.) in some areas of Kurdistan Province in Iran for the first time. Disease incidence in affected fields was approximately 100%. Symptoms were initially small, round or irregular, water-soaked spots on leaves ranging from 3 to 5 mm in diameter. These spots were limited by the leaf veins that gave them an angular appearance. Under humid conditions, tiny, white exudates formed on the undersides of the leaves and severely infected leaves turned yellow. A fluorescent pseudomonad was consistently isolated from lesions on King's medium B and characterized. Twelve strains were selected from Marivan, Dehgolan and Kamyaran in western Iran. When compared with previously identified strains, (1,2) on the basis of phenotypic, biochemical, and physiological properties, isolates were identified as Pseudomonas syringae pv. lachrymans. This was confirmed with data from whole-cell protein pattern analysis, which indicated that the strains were highly similar to reference strain 4963T (International Collection of Microorganism from Plants [ICMP]). Isolates produced round, white colonies that were 1 to 2 mm in diameter. All strains were gram negative, aerobic, levan positive, oxidase negative, potato soft rot negative, arginine dihydrolase negative, and induced a hypersensitive response on tobacco leaves. The strains were positive for catalase, urease, and 4% NaCl tolerance and negative for nitrate reduction, methyl red production, acetoin and indole production, phosphatase, gas from glucose, reducing substances from sucrose, and ketolactose tests. All strains hydrolyzed Tween 80, esculin, casein, and gelatin, but failed to hydrolyze starch and lecithin. Results for growth at 41°C and production of hydrogen sulfide from cystein and peptone were negative. In Ayers' medium, all strains produced acid from d-galactose, citrate, sucrose, raffinose, fructose, d-xylose, glucose, inositol, mannitol, sorbitol, glycerin, mannose, ribose but not from trehalose, maltose, salicin, l-rhamnose, adonitol, cellobiose, ethanol, l-sorbose, inulin, dulcitol, starch, lactose or melibiose. All strains used l-asparagine, l-lysine, aspartate and l-arginine but did not use l-tartrate, propionate, ornithine, l-tyrosine, borate, benzoate, l-tryptophan, or acetate as carbon sources. Pathogenicity of four strains was confirmed by injecting bacterial suspensions (108 CFU/ml) into the undersides of 3- to 4-week-old cucumber leaves using a sterile syringe. Sterile water was injected into cucumber leaves as a negative control. Inoculated plants were maintained in the greenhouse at 25 to 28°C with 90 to 98% relative humidity until symptoms were assessed 4 to 7 days after inoculation. Angular leaf lesions developed on inoculated cucumber. Bacterial strains were reisolated from infected tissues and confirmed as P. syringae pv. lachrymans by biochemical characterization as previously described. This disease has been observed in other areas of Iran, but to our knowledge, this is the first report that characterizes the phenotypic and biochemical properties of the bacterium and disease in Kurdistan Province.