First Report of a Leaf Spot of Radermachera sinica in China Caused by Bacillus megaterium

Radermachera sinica is widely planted as an ornamental plant in homes, offices, and malls in China. A leaf spot of R. sinica occurred in Luoyang, China, from 2013 to 2014. Lesions mostly occurred in wounds and were irregular with light brown centers and purple borders. One or more lesions on a leaf sometimes covered the entire blade. Eighty plants were surveyed in Luoyang, with disease incidence of 17%. Five millimeter pieces from the borders of lesions were surface-disinfected with 75% ethanol for 30 s, 1% sodium hypochlorite for 5 min, washed three times in sterilized distilled water, placed on nutrient agar (NA) medium at 25°C in darkness, and incubated for 24 to 48 h. Four white, round, smooth, and shiny colonies were selected for further identification. All strains were gram-positive, aerobic rods with many peritrichous flagella, and could grow in medium containing 5% NaCl. The strains were positive for catalase, starch hydrolysis, liquefaction of gelatin, reduction of nitrate, acid production from glucose, mannitol, maltose, lactose, xylose, and pectinose. The strains were positive for phenylalanine deaminase, decomposition of tyrosine, and utilization of citrate. The strains were identified by biochemical tests as Bacillus megaterium (1). To confirm pathogenicity, the strains were grown on NA for 48 h and suspended in sterile distilled water to produce a suspension with a final concentration of 108 CFU/ml. Healthy leaves of biennial R. sinica plants were sterilized with 75% ethanol and washed three times with sterilized distilled water. Fresh wounds were made with a sterile needle on the healthy leaves. Each of four strains was tested by spray inoculation with a bacterial suspension on three leaves. Sterile distilled water was used as negative control. Plants were enclosed in plastic bags and placed in a growth chamber at 28°C with 80% relative humidity. After 5 days, water-soaked lesions were observed. Two weeks later, lesions 4 mm in diameter turned light brown with purple borders, and most of lesions occurred in puncture wounds. Symptoms similar to those observed on field plants developed on all inoculated leaves, while no symptoms appeared on the control leaves. B. megaterium was re-isolated from the lesions of inoculated leaves, but not from the control leaves. To confirm the bacterial identification, PCR was performed on the 16S rDNA gene with P1/P2 (P1: CAGAGTTTGATCCTGGCT, P2: AGGAGGTGATCCAGCCGCA) (2) and 1,463 bp of the 16S rDNA gene (GenBank Accession No. KJ789369) showed 100% sequence identity to B. megaterium DSM 319 (NC_014103.1). To our knowledge, this is the first report of a leaf spot of R. sinica caused by B. megaterium in China as well as anywhere in the world. References: (1) P. Vos et al. Bergey's Manual of Systematic Bacteriology. Vol 3: The Firmicutes. Springer, 2009. (2) W. G. Weisbury et al. J. Bacteriol. 173:697, 1991.

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First Report of Bacterial Leaf Spot of Lettuce (Lactuca sativa) Caused by Xanthomonas campestris pv. vitians in Saudi Arabia

Plant Disease ◽

10.1094/pdis-93-1-0107b ◽

2009 ◽

Vol 93 (1) ◽

pp. 107-107 ◽

Cited By ~ 5

Author(s):

M. Al-Saleh ◽

Y. Ibrahim

Keyword(s):

Saudi Arabia ◽

Lactuca Sativa ◽

Leaf Spot ◽

Xanthomonas Campestris ◽

Bacterial Suspension ◽

Strictly Aerobic ◽

Distilled Water ◽

Bacterial Leaf Spot ◽

Biochemical Tests ◽

First Report

In April of 2008, lettuce (Lactuca sativa L. cv. Darkland) plants grown in the Al-Ouunia Region of Saudi Arabia were observed with numerous lesions typical of bacterial leaf spot. Leaf lesions were irregular, small, pale green to black, and 2 to 5 mm in diameter. Bacteria were isolated from diseased leaf tissues by cutting leaves into small pieces (0.5 mm) and soaking them in 2 ml of sterile distilled water. The resulting suspension was streaked onto yeast dextrose calcium carbonate agar (YDC) (1) and plates were incubated at 28°C. Large, round, butyrus, bright yellow colonies typical of Xanthomonas spp. formed after 48 h and five strains were selected for further tests. A yellow, mucoid bacterium was consistently isolated from lettuce samples with typical bacterial leaf spot symptoms. All five strains tested in this study were gram negative, oxidase negative, nitrate reduction negative, catalase and esculin hydrolysis positive, motile, and strictly aerobic. All were slightly pectolytic but not amylolytic. All were identified as Xanthomonas campestris pv. vitians. The bacterium was identified with specific oligonucleotide primers (2). This primer pair directed the amplification of an approximately 700-bp DNA fragment from total genomic DNA of all X. campestris pv. vitians strains tested. Pathogenicity tests were performed by using bacterial cultures grown on YDC for 48 h at 28°C. Each strain was suspended in sterile distilled water and the bacterial concentration was adjusted to 106 CFU/ml. Leaves of 5-week-old lettuce plants (cv. Darkland) were sprayed with the bacterial suspension. The inoculated and sterile-water-sprayed control plants were covered with polyethylene bags for 48 h at 25°C, after which the bags were removed and plants were transferred to a greenhouse at 25 to 28°C (1). All strains were pathogenic on the lettuce cv. Darkland, causing typical bacterial leaf spot symptoms by 2 weeks after inoculation. All inoculated plants showed typical symptoms of bacterial leaf spot and symptoms similar to those observed on the samples collected. No symptoms developed on the control plants. The bacterium was reisolated from inoculated plants and identified as X. campestris pv. vitians by morphological, physiological, and biochemical tests as described above. To our knowledge, this is the first report of bacterial leaf spot of lettuce by X. campestris pv. vitians in Saudi Arabia. References: (1) F. Sahin and A. Miller. Plant Dis.81:1443, 1997. (2) J. D. Barak. Plant Dis.85:169, 2001.

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First Report of Leaf Spot of Dracaena reflexa Caused by Burkholderia gladioli Worldwide

Plant Disease ◽

10.1094/pdis-94-6-0781c ◽

2010 ◽

Vol 94 (6) ◽

pp. 781-781

Author(s):

M. K. Romberg ◽

R. L. Griffin ◽

S. Murugan ◽

B. D. Quinn ◽

B. J. R. Alexander

Keyword(s):

16S Rdna ◽

Leaf Spot ◽

Natural Infection ◽

Similarity Index ◽

Leading Edge ◽

Bacterial Suspension ◽

Distilled Water ◽

Carbon Utilization ◽

First Report ◽

Burkholderia Gladioli

In December 2008 (austral summer), a new disease of Dracaena reflexa Lam. cv. Anita was observed in a postentry quarantine greenhouse near Auckland, New Zealand on plants imported from Costa Rica. Symptoms included rust-colored, water-soaked lesions with chlorotic margins approximately 5 by 10 mm. When the disease was first noticed, incidence approached 80%, but subsequent reduction in greenhouse temperature dramatically reduced symptom expression and lesions were only visible on some leaf tips. Bacteria consistently isolated from the lesions on King's medium B (KB) were cream-colored, shiny, and produced a yellow, diffusible, nonfluorescent pigment. All isolates were able to rot onion slices. On the basis of BIOLOG (Hayward, CA) carbon utilization profiles, isolates were initially identified as Burkholderia gladioli (Severini 1913) Yabuuchi et al. 1993 with a probability index of 100% and a similarity index of 0.85. For molecular identification, a near full-length sequence of the 16S rDNA gene was amplified from all isolates using primers fD2 and rP1 (1), obtaining a PCR product of approximately 1,500 bp. The nucleotide sequences were 100% identical to a number of B. gladioli GenBank entries, including Accession Nos. EF193645 and EF088209. To confirm pathogenicity, three isolates (two isolated prior to greenhouse temperature reduction and one after) were used. Three D. reflexa plants were inoculated per bacterial isolate by wounding three young fully expanded leaves on each plant (four wounds per leaf) and spraying the leaves with a bacterial suspension in sterile distilled water at 108 CFU/ml. At the same time, Gladiolus nanus plants were inoculated in a similar manner. Control plants (D. reflexa and G. nanus) were wounded and sprayed with sterile distilled water. All inoculated plants were covered with plastic bags to maintain humidity and placed in a growth chamber at 25°C. At 3 days, all inoculated plants began to show water soaking and reddish coloration around the inoculation sites, and by 7 days, the lesions had expanded to resemble natural infection. Bacteria isolated on KB from the leading edge of each lesion were morphologically identical to the initial isolates. No bacteria were recovered from the wound sites on the control plants. The 16S rDNA sequences of selected isolates from inoculated plants showed 100% identity to the sequences of the initial isolates, thereby fulfilling Koch's postulates. To our knowledge, this is the first report of B. gladioli causing leaf spot of D. reflexa in the world. Reference: (1) W. G. Weisburg et al. J. Bacteriol. 173:697, 1991.

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First Report of a Soft Rot of Philodendron ‘Con-go’ in China Caused by Dickeya dieffenbachiae

Plant Disease ◽

10.1094/pdis-09-11-0795 ◽

2012 ◽

Vol 96 (3) ◽

pp. 452-452 ◽

Cited By ~ 5

Author(s):

B. R. Lin ◽

H. F. Shen ◽

J. N. Zhou ◽

X. M. Pu ◽

Z. N. Chen ◽

...

Keyword(s):

16S Rdna ◽

Phylogenetic Trees ◽

Soft Rot ◽

Gyrb Gene ◽

Control Treatment ◽

Gene Sequences ◽

16S Rdna Gene ◽

First Report ◽

Rdna Gene ◽

Plastic Bags

Philodendron is a popular foliage plant cultivated in interiorscapes of homes, offices, and malls throughout China. A severe outbreak of a soft rot of Philodendron ‘Con-go’ occurred in Guangzhou, China from 2010 to 2011. The disease was characterized by leaf infections starting as pinpoint spots that are water soaked and yellow to pale brown. The lesions are sometimes surrounded by a diffuse yellow halo. When the humidity is high and temperatures are warm to hot, the spots expand rapidly, becoming slimy, irregular, and sunken with light tan centers, darker brown borders, and diffused yellow margins and may involve the entire leaf in a few days. An invasion of the midrib and larger veins by the causal bacterium often results in advancement into the petiole and stem. A survey of three areas of production of Philodendron ‘Con-go’ (5 ha) in Guangzhou revealed that 91% of the fields were affected at an incidence ranging from 15 to 30%. Of 41 bacterial isolates obtained from lesions, three were selected randomly for further characterization. All strains were gram negative, negative for oxidase and positive for catalase and tryptophanase (indole production), and utilized citrate, tartrate, malonate, glucose, sucrose, fructose, and maltose but not glucopyranoside, trehalose, or palatinose. Biolog analysis (version 4.20.05, Hayward, CA) identified the isolates as Pectobacterium chrysanthemi (SIM 0.804 to 0.914). According to Samson et al. (1), it was renamed as a Dickeya sp. PCR was performed on the 16S rDNA gene with primers 27f and 1495r (3) and 1,423 bp of the 16S rDNA gene (GenBank No. JN709491) showed 99% identity to P. chrysanthemi (GenBank No. AF373202), and 98% to Dickeya dieffenbachiae (GenBank No. JF311644). Additionally, the gyrB gene was amplified with primers gyrB-f1 (5′-atgtcgaattcttatgactcctc-3′) and gyrB-r1 (5′-tcaratatcratattcgcygctttc-3′) designed based on all the submitted gyrB gene sequences of Dickeya spp. The dnaX gene was amplified with primers dnaXf and dnaXr (2). The products were sequenced and phylogeny analyses were performed by means of MEGA 5.05. Results showed that the gyrB and the dnaX genes of the strains were 98% homologous to those of D. dieffenbachiae (GenBank Nos. JF311652 and GQ904757). Therefore, on the basis of phylogenetic trees of the 16S rDNA, gyrB, and dnaX gene sequences, the bacterial isolate named PC1 is related to D. dieffenbachiae (100% bootstrap values). Pathogenicity of each of the three strains on Philodendron ‘Con-go’ was confirmed by injecting 60 50-day-old seedlings each with 0.1 ml of the isolate suspension (108 CFU/ml) into the leaves. Another 60 were injected with sterile water to serve as the control treatment. Plants were enclosed in plastic bags and returned to the greenhouse under 50% shade at 32°C day and 28°C night temperatures with high humidity. After 72 h, all the injected plants started to show symptoms similar to those observed on field plants, but no symptoms appeared on the control plants. The reisolates were identical to the inoculated strains in biochemical characteristics. Bacteria characteristic of the inoculated strains were not reisolated from the control plants. To our knowledge, this is the first report of D. dieffenbachiae causing soft rot of Philodendron ‘Con-go' in China. References: (1) R. Samson et al. Evol. Microbiol. 55:1415, 2005. (2) M. Sławiak et al. Eur. J. Plant Pathol. 125:245, 2009. (3) W. G. Weisbury et al. J. Bacteriol. 173:697, 1991.

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First Report of Bacterial Leaf Spot of Coriander Caused by Pseudomonas syringae pv. coriandricola in China

Plant Disease ◽

10.1094/pdis-09-21-1901-pdn ◽

2021 ◽

Author(s):

Lei Li ◽

Yishuo Huang ◽

Yanxia Shi ◽

A LI CHAI ◽

Xuewen Xie ◽

...

Keyword(s):

Pseudomonas Syringae ◽

Leaf Spot ◽

Citrate Synthase ◽

Morphological Characteristics ◽

Likelihood Method ◽

Bacterial Suspension ◽

Leaf Spot Disease ◽

Distilled Water ◽

Bacterial Leaf Spot ◽

First Report

Coriander (Coriandrum sativum L.) or Chinese parsley is a culinary herb with multiple medicinal effects that are widely used in cooking and traditional medicine. From September to November 2019, symptoms were observed in 2-month-old coriander plants from coriander ﬁelds in Lanzhou and Wenzhou, China. The disease developed rapidly under cold and wet climatic conditions, and the infection rate was almost 80% in open coriander fields. Typical symptoms on leaves included small, water-soaked blotches and irregular brown spots surrounding haloes; as the disease progressed, the spots coalesced into necrotic areas. Symptomatic leaf tissue was surface sterilized, macerated in sterile distilled water, and cultured on nutrient agar plates at 28 °C for 48 h (Koike and Bull, 2006). After incubation, six bacterial colonies, which were individually isolated from collected samples from two different areas, were selected for further study. Colonies on NA plate were small, round, raised, white to cream-colored, and had smooth margins. All bacterial isolates were gram-negative, rod-shaped and nonfluorescent on King's B medium. The bacteria were positive for levan production, Tween 80 hydrolysis, and tobacco hypersensitivity but negative for oxidase, potato slice rot test, arginine dihydrolase, ice nucleation activity, indole production and H2S production. The suspension of representative isolate for inoculating of plants was obtained from single colony on King's B medium for 2-3 days at 28 °C. DNA was extracted from bacterial suspensions of YS2003200102 cultured in 20 ml of King’s B medium broth at 28 °C for 1 day. Extraction was performed with a TIANamp Bacterial DNA Kit (TIANGEN, China) according to the manufacturer’s recommendations. The pathogen was confirmed by amplification and sequencing of the glyceraldehyde-3-phosphate dehydrogenase A (gapA) gene, the citrate synthase (gltA) gene, the DNA gyrase B (gyrB) gene and the RNA polymerase sigma factor 70 (rpoD) gene using gapA-For/gapA-Rev, gltA-For/gltA-Rev, gyrB-For/gryB-Rev, rpoD-For/rpoD-Rev primers, respectively (Popović et al., 2019). The sequences of the PCR products were deposited in GenBank with accession numbers MZ681931 (gapA), MZ681932 (gltA), MZ681933 (gyrB), and MZ681934 (rpoD). Phylogenetic analysis of multiple genes (Xu and Miller, 2013) was conducted with the maximum likelihood method using MEGA7. The sequences of our isolates and ten published sequences of P. syringae pv. coriandricola were clustered into one clade with a 100% confidence level. To confirm the pathogenicity of isolate YS2003200102, 2-month-old healthy coriander plants were inoculated by spraying the leaves with a bacterial suspension (108 CFU ml−1) at 28 °C incubation temperature and 70% relative humidity condition, and sterile distilled water was applied as a negative control treatment (Cazorla et al. 2005). Three replicates were conducted for every isolate, and each replicate included 6 coriander plants. After twelve days, only the inoculated leaves with bacterial suspension showed bacterial leaf spot resembling those observed on naturally infected coriander leaves. Cultures re-isolated from symptomatic leaves showed the same morphological characteristics and molecular traits as those initially isolated from infected leaves in the field. This bacterium was previously reported causing leaf spot of coriander in India and Spain (Gupta et al. 2013; Cazorla et al. 2005). To our knowledge, this is the first report of P. syringae pv. coriandricola causing leaf spot disease on coriander in China. Studies are needed on strategies to manage P. syringae pv. coriandricola in crops, because its prevalence may cause yield loss on coriander in China.

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First Report of a Chaetomella sp. Causing a Leaf Spot on Sansevieria trifasciata in China

Plant Disease ◽

10.1094/pdis-10-12-0993-pdn ◽

2013 ◽

Vol 97 (7) ◽

pp. 992-992 ◽

Cited By ~ 4

Author(s):

Y. L. Li ◽

Z. Zhou ◽

W. Lu ◽

J. R. Ye

Keyword(s):

Leaf Spot ◽

Disease Incidence ◽

Morphological Characteristics ◽

Leaf Tissue ◽

Aerial Mycelium ◽

Control Treatment ◽

Distilled Water ◽

First Report ◽

Potted Plants ◽

Fungal Isolates

Sansevieria trifasciata originates from tropical West Africa. It is widely planted as a potted ornamental in China for improving indoor air quality (1). In February 2011, leaves of S. trifasciata plants in an ornamental market of Anle, Luoyang City, China, were observed with sunken brown lesions up to 20 mm in diameter, and with black pycnidia present in the lesions. One hundred potted plants were examined, with disease incidence at 20%. The symptomatic leaves affected the ornamental value of the plants. A section of leaf tissue from the periphery of two lesions from a plant was cut into 1 cm2 pieces, soaked in 70% ethanol for 30 s, sterilized with 0.1% HgCl2 for 2 min, then washed five times in sterilized distilled water. The pieces were incubated at 28°C on potato dextrose agar (PDA). Colonies of two isolates were brown with submerged hyphae, and aerial mycelium was rare. Abundant and scattered pycnidia were reniform, dark brown, and 200 to 350 × 100 to 250 μm. There were two types of setae on the pycnidia: 1) dark brown setae with inward curved tops, and 2) straight, brown setae. Conidia were hyaline, unicellular, cylindrical, and 3.75 to 6.25 × 1.25 to 2.50 μm. Morphological characteristics suggested the two fungal isolates were a Chaetomella sp. To confirm pathogenicity, six mature leaves of a potted S. trifasciata plant were wounded with a sterile pin after wiping each leaf surface with 70% ethanol and washing each leaf with sterilized distilled water three times. A 0.5 cm mycelial disk cut from the margin of a 5-day-old colony on a PDA plate was placed on each pin-wounded leaf, ensuring that the mycelium was in contact with the wound. Non-colonized PDA discs were placed on pin-wounded leaves as the control treatment. Each of two fungal isolates was inoculated on two leaves, and the control treatment was done similarly on two leaves. The inoculated plant was placed in a growth chamber at 28°C with 80% relative humidity. After 7 days, inoculated leaves produced brown lesions with black pycnidia, but no symptoms developed on the control leaves. A Chaetomella sp. was reisolated from the lesions of inoculated leaves, but not from the control leaves. An additional two potted plants were inoculated using the same methods as replications of the experiment, with identical results. To confirm the fungal identification, the internal transcribed spacer (ITS) region of rDNA of the two isolates was amplified using primers ITS1 and ITS4 (2) and sequenced. The sequences were identical (GenBank Accession No. KC515097) and exhibited 99% nucleotide identity to the ITS sequence of an isolate of Chaetomella sp. in GenBank (AJ301961). To our knowledge, this is the first report of a leaf spot of S. trifasciata caused by Chaetomella sp. in China as well as anywhere in the world. References: (1) X. Z. Guo et al. Subtropical Crops Commun. Zhejiang 27:9, 2005. (2) T. J. White et al. PCR Protocols: A Guide to Methods and Applications. M. A. Innis et al., eds. Academic Press, San Diego, CA, 1990.

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First Report of Bacterial Spot Caused by Xanthomonas campestris pv. vesicatoria on Sweet Pepper (Capsicum annuum L.) in Saudi Arabia

Plant Disease ◽

10.1094/pdis-04-12-0354-pdn ◽

2012 ◽

Vol 96 (11) ◽

pp. 1690-1690 ◽

Cited By ~ 1

Author(s):

Y. Ibrahim ◽

M. Al-Saleh

Keyword(s):

Saudi Arabia ◽

Xanthomonas Campestris ◽

Sweet Pepper ◽

Negative Control ◽

Bacterial Suspension ◽

Distilled Water ◽

Bacterial Spot ◽

Biochemical Tests ◽

First Report ◽

Pepper Fruit

In the summer of 2009 and 2010, 18 sweet pepper fruit with blister-like, raised, rough lesions were collected from four greenhouses (total of 0.1 ha) in the Al-Kharj region of Saudi Arabia. All samples were collected from commercial crops of the sweet pepper cv. California Wonder. Disease incidence was ≤5%. Isolations were made from all diseased fruits. A small piece (3 mm2) of symptomatic tissue from pepper fruit was placed in a sterile mortar and macerated in sterile distilled water with a pestle. A loopful of bacterial suspension from each sample was streaked onto Tween B agar medium (3). Plates were incubated at 28°C for 48 h. Single yellow, circular, butyrous, shiny colonies were picked from the plates and transferred to nutrient agar plates containing 5% D+ glucose agar (NGA). Gram-negative, rod-shaped bacteria were consistently isolated from the fruit and 10 of the isolates were identified as Xanthomonas campestris pv. vesicatoria on the basis of morphological, physiological, and biochemical tests (1,2). The isolates were oxidase positive and levan negative, arginine-dihydrolase positive, and did not macerate potato discs. The isolates were also non-fluorescent, grew at 37 and 4°C but not at 40°C, did not liquefy gelatine or starch, but did produce H2S. The identity of the 10 bacterial strains was confirmed by PCR assay using primers RST65 and RST69 (4). Four-week old pepper plants (cv. California Wonder) were inoculated by spraying five potted plants with each isolate using a bacterial suspension (108 CFU/ml). Sterile distilled water was sprayed on an additional five plants as a negative control treatment. The bacterial isolates caused necrotic lesions, each with a yellow halo, on leaves of inoculated plants. Bacteria reisolated from the necrotic lesions using the technique previously described were identical to the original strains according to the morphological, cultural, and biochemical tests described above. Negative control plants inoculated with sterile distilled water did not show symptoms and no bacterial colonies were recovered from them. To our knowledge, this is the first report of bacterial spot on pepper fruits in Saudi Arabia. References: (2) R. F. Bradbury. Genus II Xanthomonas Dowson 1939. In: Bergey's Manual of Systematic Bacteriology, Vol. 1, Krieg, R., Holt, J. G. (Eds.), Williams & Wilkins Co., Baltimore, MD, 1987. (3) R. A. Lelliott and D. E. Stead. Methods for the Diagnosis of Bacterial Diseases of Plants. Blackwell Scientific Publications, Oxford, UK. (1) R. G. McGuire et al. Plant Dis 70:887, 1986. (4) A. Obradovic et al. Eur. J. Plant Pathol. 110:285, 2004.

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First Report of a γ 3-Proteobacterium Associated with Diseased Strawberries in Italy

Plant Disease ◽

10.1094/pdis-91-12-1688b ◽

2007 ◽

Vol 91 (12) ◽

pp. 1688-1688 ◽

Cited By ~ 7

Author(s):

F. Terlizzi ◽

A. R. Babini ◽

C. Lanzoni ◽

A. Pisi ◽

R. Credi ◽

...

Keyword(s):

16S Rdna ◽

16S Rdna Gene ◽

First Report ◽

Genome Region ◽

Rdna Gene ◽

Rdna Sequences ◽

Pcr Products ◽

Emilia Romagna Region ◽

Young Leaves ◽

Stolbur Phytoplasma

During the fall seasons of 2005 and 2006, diseased strawberry plants (Fragaria × ananassa Duch.) were observed in nurseries and production fields in Ferrara, Forli-Cesena, and Ravenna provinces (Emilia-Romagna region, northern Italy). Symptoms consisted of a conspicuous plant stunting with a poor root system. Older leaves rolled upward and displayed a marked premature purplish discoloration, while young leaves were cupped, chlorotic, generally reduced in size, and had shortened petioles. This strawberry disorder was similar to “marginal chlorosis”, an infectious disease occurring in France that can be induced by two different phloem-limited uncultured bacteria: the γ 3-proteobacterium ‘Candidatus Phlomobacter fragariae’ and the stolbur phytoplasma (16SrXII-A). In strawberry production fields, ‘Ca. P. fragariae’ is reported as being the prevalent agent of this disease (1). Sixty-seven diseased plants were collected from production fields and nurseries for testing for ‘Ca. P. fragariae’. Leaf samples were analyzed by 4′,6-diamidine-2-phenylindole staining and PCR. Forty samples showed fluorescent DNA in the phloem, whereas no fluorescence was observed in symptomless strawberries. When tested by PCR with primers Fra4/Fra5, which amplify a 550-bp fragment of the 16S rDNA region of ‘Ca. P. fragariae’ (1), 13 of 36 strawberries from production fields and 1 of 31 nursery plants gave a positive reaction. On the other hand, 21 samples from nurseries and 5 from production fields tested positive for stolbur phytoplasma (3). No amplification was obtained with DNA from symptomless or healthy strawberry plants. Sequencing Fra4/Fra5 amplicons from three samples (GenBank Accession Nos. DQ362916–DQ362918) showed a 98.1 to 98.6% and a 98.3 to 98.8% identity with the published sequences of the French isolate “LG2001” (GenBank Accession No. AM110766) and the Japanese isolate J-B (GenBank Accession No. AB246669) of ‘Ca. P. fragariae’, respectively. Higher homology (99.2 to 99.8%) was found with another bacterium-like organism (BLO) of the γ 3-proteobacteria subgroup (GenBank Accession No. AY057392) associated with the syndrome “basses richesses” of sugar beet (SBR). Furthermore, PCR assays performed with primers Pfr1/Pfr4, specific for spoT gene of ‘Ca. P. fragariae’, did not show any amplification with DNA from the 14 diseased strawberry plants tested. This is in agreement with the SBR BLO identification (2). To better characterize the Italian isolates, the full-length 16S rDNA gene was analyzed with primers fd1/Fra4 and Fra5/rp1, which amplify the 5′ and 3′ region of 16S rDNA gene of the proteobacteria, respectively (2). PCR products from eight isolates were sequenced, and the 16S rDNA sequences obtained (GenBank Accession Nos. DQ538372–DQ538379) showed a 96.4 to 97.3% identity with the known ‘Ca. P. fragariae’ isolates, while a higher homology (99.4 to 99.9%) was again found with the SBR BLO. To our knowledge, this is the first report of a γ 3-proteobacterium affecting strawberry in Italy. In the genome region analyzed, our isolates are more similar to the SBR BLO than to ‘Ca. P. fragariae’. Further work is in progress to investigate incidence, geographical distribution, epidemiology, and host range of this pathogen in Italy. References: (1) J. L. Danet et al. Phytopathology 93:644, 2003. (2) O. Semetey et al. Phytopathology 97:72, 2007. (3) F. Terlizzi et al. Plant Dis. 90:831, 2006.

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First Report of Crown Gall, Caused by Agrobacterium tumefaciens on Soapberry (Sapindus delavayi) in China

Plant Disease ◽

10.1094/pdis-10-12-0988-pdn ◽

2013 ◽

Vol 97 (5) ◽

pp. 685-685

Author(s):

Y. J. Wang ◽

Y. Y. He ◽

Z. Xie ◽

L. Q. Zhang

Keyword(s):

Agrobacterium Tumefaciens ◽

16S Rdna ◽

Crown Gall ◽

Maximum Parsimony Analysis ◽

Bacterial Suspension ◽

Identification System ◽

Distilled Water ◽

Bacterial Identification ◽

Poor Growth ◽

First Report

Soapberry (Sapindus delavayi (Franch.) Radlk.,) plants are widely grown as shade trees in the subtropical to tropical regions of China. In July 2011, large, aerial galls were observed on the above-ground trunks of 5-year-old soapberry plants in two commercial nursery gardens located in Zhejiang Province. Disease incidence was estimated to be 75%. The galls varied in weight from 2 to 24 g and in texture from soft and spongy to hard, and in some cases, the galls completely girdled the trunk. The trees with galls exhibited poor growth compared with healthy trees. Isolations from the grinded and macerated galls yielded nearly pure white, circular, and glistening bacterial colonies on Roy Sauer medium (2). Six random colonies from different galls were selected for bacterial identification, and showed the same morphological, physiological, and biochemical characters and 16S rDNA sequences. All six isolates (isolate SD01 to SD06) were gram negative, rod-shaped bacteria. Carbon source utilization testing with the Biolog GN Bacterial Identification System (version 3.50) confirmed the bacteria as Agrobacterium tumefaciens with a similarity of 0.90. The most-parsimonious tree from the maximum parsimony analysis (PHYLIP package, version 3.68, 500 replicates) of bacterial 16S rDNA gene sequences showed that A. tumefaciens SD01 (GenBank Accession No. JX997939) clustered phylogenetically most closely (99.5% similarity) with A. tumefaciens C58 (AE007870.2). Pathogenicity was confirmed by injecting 3- to 5-week old tomato and sunflower plants and 2-year-old soapberry with approximately 5 μl of the bacterial suspension (108 CFU/ml) in sterile, distilled water. Sterile distilled water was used as a negative control. Ten plants of each treatment were inoculated. Inoculated plants were then transferred to a greenhouse at 25°C. Typical tumors developed at the inoculation sites on tomatoes and sunflower plants 3 weeks after inoculation and on soapberry 6 weeks after inoculation. No symptoms were observed on the control plants. The bacteria that were readily reisolated from the inoculated plants exhibited the same morphological, physiological characters and 16S rDNA sequence as the original culture and were confirmed as A. tumefaciens, fulfilling Koch's postulates. A. tumefaciens is endemic to China and has a very wide host range (1). However, crown gall of soapberry has never been found in China and other countries. To our knowledge, this is the first report of A. tumefaciens on soapberry plants in China. References: (1) M. A. Escobar and A. M. Dandekar. Trends Plant Sci. 8:380, 2003. (2) L. W. Moore et al. Page 17 in: Laboratory Guide for Identification of Plant Pathogenic Bacteria. 3rd ed. N. W. Schaad et al., eds. The American Phytopathological Society, St. Paul, MN, 2001.

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First Report of Leaf Blight on Saposhnikovia divaricata by Pseudomonas viridiflava in Gansu, China

Plant Disease ◽

10.1094/pdis-06-14-0641-pdn ◽

2015 ◽

Vol 99 (2) ◽

pp. 281-281 ◽

Cited By ~ 1

Author(s):

Y. Wang ◽

C. Y. Zeng ◽

X. R. Chen ◽

C. D. Yang

Keyword(s):

16S Rdna ◽

Leaf Blight ◽

Gansu Province ◽

Rdna Sequence ◽

Bacterial Suspension ◽

Distilled Water ◽

16S Rdna Sequence ◽

First Report ◽

Saposhnikovia Divaricata ◽

The Impact

Saposhnikovia divaricata (Turcz) Schischk, a perennial plant in the Umbelliferae, is widely cultivated in north China. As a traditional Chinese medicine, it can be used to cure colds and rheumatism (1). During disease surveys on medicinal plants in August 2010, a bacterial leaf blight was discovered with a general incidence of 40 to 60% on S. divaricata farms in Longxi, Weiyuan County in Gansu China. In young plants, tiny yellow-white points were visible on the backs of the leaves. They then expanded to 2- to 3-mm oil-soaked lesions; leaves appeared crimped and deformed. Later the leaves shriveled; black-brown oil-soaked lesions appeared on the vein and the tissue around it; and black streaks appeared on the stems. Ten diseased leaf and stem tissues were cut into 4- to 5-mm squares, surface-sterilized in 1% sodium hypochlorite for 1 min, rinsed three times, and macerated for 5 min in sterilized distilled water. They were then streaked onto nutrient agar (NA) medium and incubated at 28°C for 3 days. Colonies on NA were round, smooth, translucent, and yellowish green. They were Gram negative and induced a hypersensitive response on tobacco (Nicotiana tabacum L.) leaves. The strain was positive for gelatin, catalase, oxidase, and utilization of glucose and saccharose. Pathogenicity tests were performed by spraying bacterial suspension containing 107 CFU/ml on six leaves of three healthy potted S. divaricata plants and injecting it into another six leaves on three plants. Plants inoculated with sterile distilled water alone served as controls. They were placed in a growth chamber at 25°C and bagged for 24 h to maintain >95% humidity. Thirty-six hours after inoculation, the inoculated leaves appeared water-soaked; 10 days later, the symptoms were apparent on leaves and the plant wilted. The negative control appeared normal. Finally, Koch's postulates were verified by re-isolating P. viridiflava from the leaves with typical blight. The genomic DNA of the isolate was extracted, and the partial 16S rDNA sequence was amplified with a universal bacterial primer set (27f and 1492r) (2). The sequence was deposited in GenBank as KM030291. BLAST search yielded 99% identity with P. viridiflava strains, including the strains KNOX209 (AY604847), RMX3.1b (AY574911), ME3.1b (AY574909), and UASWS0038 (AY919300). Based on the symptoms, colony morphology, biochemical tests, and 16S rDNA sequence identity, the pathogen was identified as P. viridiflava. To our knowledge, this is the first report of leaf blight of S. divaricata by P. viridiflava in Gansu province of China. In Jilin province, the same disease was reported in 2008 (3). The impact of P. viridiflava on S. divaricata production is not yet known. References: (1) Committee of China Pharmacopoeia. Pharmacop. People's Repub. 1:102, 2005. (2) C. Morenol et al. Microbiology 148:1233, 2002. (3) W. Xue. Dissertation. Jilin Agric. Univ. 1, 2008.

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First Report of Bacterial Bark Canker of Walnut Caused by Brenneria nigrifluens in Hungary

Plant Disease ◽

10.1094/pdis-09-13-0949-pdn ◽

2014 ◽

Vol 98 (7) ◽

pp. 988-988 ◽

Cited By ~ 2

Author(s):

A. Végh ◽

A. Tóth ◽

Á. Zámbó ◽

G. Borsos ◽

L. Palkovics

Keyword(s):

16S Rdna ◽

Juglans Regia ◽

Negative Control ◽

Bacterial Suspension ◽

Lake Balaton ◽

Biochemical Tests ◽

First Report ◽

Potted Plants ◽

Sequence Identity ◽

Plastic Bags

During August 2012, vertical oozing cankers were sporadically observed on trunks and branches of walnut trees (Juglans regia) in the city of Zánka, near Lake Balaton and other parts of Hungary including Budapest, Győr, and Tatabánya cities. Cankers were observed on trunks and branches where brownish-black exudates staining the bark appeared mainly in the summer. Isolations were performed primarily from exudates but also from infected tissues using King's medium B (KB) (3) and EMB medium (2). Colonies similar in appearance to Brenneria nigrifluens (syn.: Erwinia nigrifluens) (1,5) were isolated. The bacterium, first reported in California, was also recorded in Iran, Spain, France, and several Italian locations, on walnut trees. The bacterial strain was gram negative and did not induce a hypersensitive response on tobacco (Nicotiana tabacum L. ‘White Burley’) leaves. The bacterium grew at 26°C. Colonies on KB were white and non-fluorescent, but on EMB medium were a typical dark purple with metallic green sheen. The results of substrate utilization profiling using the API 20E kit (Biomérieux, Marcy l'Etoile, France) showed that the bacterium belonged to the Enterobacteriaceae. The strain was positive for citrate utilization, H2S, and acetoin production and urease, glucose, inositol, saccharose, and arabinose reactions. Pathogenicity was tested by injecting five young healthy walnut branches on two separate 2-year-old grafted potted plants with a bacterial suspension containing 107 CFU/ml. Negative controls were walnut branches injected with sterile distilled water. Branches were enclosed in plastic bags and incubated in a greenhouse under 80% shade at 26°C day and 17°C night temperatures. Three months after inoculation, necrotic lesions were observed in the inner bark and dark lines were observed in internal wood, but no external cankers were observed on inoculated branches. The negative control appeared normal. B. nigrifluens was re-isolated from lesions on inoculated branches and identified as described above; thus, Koch's postulates were fulfilled. For molecular identification of the pathogen, 16S rDNA amplification was performed using genomic DNA from strain Bn-WalnutZa-Hun1 with a universal bacterial primer set (63f and 1389r) (4). The PCR products were cloned into a pGEM T-Easy vector (Promega, Madison, WI) and transformed into Escherichia coli DH5α cells. A recombinant plasmid (2A2.5) was sequenced using M13 forward and reverse primers. The sequence was deposited in NCBI GenBank (Accession No. HF936707) and showed 99% sequence identity with a number of B. nigrifluens strains, including type strains Z96095.1, AJ233415.1, JX484740.1, JX484739.1, JX484738.1, and FJ611884.1. On the basis of the symptoms, colony morphology, biochemical tests, and 16S rDNA sequence identity, the pathogen was identified as Brenneria nigrifluens. To our knowledge, this is the first report of a natural outbreak of bacterial bark canker on walnut in Hungary and the presence of the pathogen may seriously influence in local orchards and garden production in the future. References: (1) L. Hauben et al. Appl Microbiol 21:384, 1998. (2) J. E. Holt-Harris and O. Teague. J. Infect. Dis. 18:596, 1916. (3) E. O. King et al. J. Lab. Clin. Med. 44:301, 1954. (4) A. M. Osborn et al. Environ. Microbiol. 2:39, 2000. (5) E. E. Wilson et al. Phytopathology 47:669, 1957.

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