First Report of Leaf Spot Disease of Maize Caused by Pantoea ananatis in Argentina

From 2007 to 2008, an uncharacterized disease of maize (Zea mays L.) was observed in commercial fields of Laguna Blanca, Formosa, Argentina and from different fields of Santa Fe and Catamarca provinces of Argentina. Symptoms included light-colored necrotic streaks on leaves and tan or white irregular blotches that sometimes were surrounded by reddish purple-to-dark brown margins. Severity of symptoms varied greatly from one field to another. Abundant bacterial streaming was observed from lesions when examined at ×150. Gram-negative, facultatively anaerobic bacteria were consistently isolated from lesions. These formed light yellow-to-orange, glistening, convex colonies on yeast dextrose calcium carbonate agar incubated at 30°C. Ten isolates from ten different symptomatic plants were selected for further study. All isolates were motile, induced a hypersensitive response in tobacco plants, and were oxidase negative. Colonies developed at 37°C. Physiological and biochemical characterization with the API 20E test strips and database (bioMerieux, Buenos Aires, Argentina) showed that the strains belonged to the genus Pantoea. All strains were positive for β-galactosidase, utilized citrate and tartrate, and produced acid from d-glucose, d-mannitol, d-melibiose, l-arabinose, sucrose, meso-inositol, glycerol, d-sorbitol, and amygdalin. All were negative for arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase, tryptophane deaminase, H2S production, urease, and reduction of nitrate to nitrite. Variable results were obtained for indole, gelatinase, and l-rhamnose. Their identity was confirmed by sequencing the 16S rRNA gene strain F327 (GenBank Accession No. GU068363). A BlastN search of GenBank revealed 99% nt identity with strains LMG 20103 (AF364847.1), LMG 20105 (AF364845.1), and LMG 2665 (FJ611815.1) of Pantoea ananatis. Pathogenicity was verified on Z. mays (EM 6079 HX, Dow Morgan) by injection-infiltration of bacterial suspensions at 105 CFU/ml. Controls were infiltrated with sterile distilled water. Plants were kept at 26 ± 3°C in a greenhouse. Symptoms were first detected 15 to 17 days after inoculation and then lesions expanded to resemble natural infections within 30 days. Bacteria were reisolated and the original and reisolated strains were compared by using repetitive sequence-based (rep)-PCR with ERIC primers (1) and fingerprints of the reisolated strains were identical to those of the original strains, thereby fulfilling Koch's postulates. No lesions were observed on controls. Known strains of P. stewartii from the United States (SW2, DC400, DC441, and DC283) were also tested for comparison. On the basis of sequencing data, pathogenicity, and physiological tests, the pathogen was identified as P. ananatis (4). To our knowledge, this is the first report of P. ananatis causing a disease of maize in Argentina, although a similar disease has been reported in Brazil (2) and Mexico (3). References: (1) F. J. Louws et al. Appl. Environ. Microbiol. 60:2286, 1994. (2) L. D. Paccola-Meirelles et al. J. Phytopathol. 149:275, 2001. (3) R. Pérez-y-Terrón et al. Australas. Plant Dis. Notes 4:96, 2009. (4) N. W. Schaad et al., eds. Laboratory Guide for Identification of Plant Pathogenic Bacteria. 3rd ed. The American Phytopathological Society, St. Paul, MN, 2001.

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First Report of Bacterial Soft Rot on Vanda Orchids Caused by Dickeya chrysanthemi (Erwinia chrysanthemi) in the United States

Plant Disease ◽

10.1094/pdis-92-6-0977a ◽

2008 ◽

Vol 92 (6) ◽

pp. 977-977 ◽

Cited By ~ 6

Author(s):

R. A. Cating ◽

J. C. Hong ◽

A. J. Palmateer ◽

C. M. Stiles ◽

E. R. Dickstein

Keyword(s):

Pathogenic Bacteria ◽

Soft Rot ◽

The United States ◽

Erwinia Chrysanthemi ◽

Disease Spread ◽

Rrna Gene ◽

Bacterial Disease ◽

Water Control ◽

First Report ◽

Microbial Identification

Vanda orchids are epiphytes grown for their attractive flowers by commercial producers and hobbyists throughout Florida. In August 2007, five Vanda hybrids, with an economic value of $150 each, were found at a nursery in central Florida with leaves that were macerated, brown, and water soaked. According to the growers, the plants were normal the previous day but symptoms developed rapidly. The plants were immediately removed from the greenhouse to prevent potential disease spread. Bacteria were isolated according to the method of Schaad et al. (1). Isolated bacteria grew at 37°C, were gram negative, degraded pectate, and produced phosphatase. MIDI (Sherlock version TSBA 4.10; Microbial Identification 16 System, Newark, DE) (SIM 0.906) identified the bacteria as Erwinia chrysanthemi (Dickeya chrysanthemi Burkholder et al. 1953) Samson et al. 2005. PCR was performed on the 16S rRNA gene (GenBank Accession No. EU526397) with primers 27f (5′-GAGAGTTTGATCCTG GCTCAG-3′) and 1495r (5′-TACGGCTACCTTGTTACGA-3′) (2). Subsequent DNA sequencing and GenBank search showed the isolated strain is 99% identical to that of Dickeya chrysanthemi. Four leaves each of six Vanda hybrids were inoculated by injecting approximately 150 μl of a bacteria suspension at 1 × 108 CFU/ml into each leaf. One plant was inoculated with water in each of four leaves. Plants were enclosed in plastic bags and returned to the greenhouse under 50% shade at 29°C day and 17°C night temperatures. Within 24 h, soft rot symptoms appeared on inoculated leaves. The water control appeared normal. D. chrysanthemi was reisolated and identified with the above method, thus Koch's postulates were fulfilled. To our knowledge, this is the first report of a soft rot caused by D. chrysanthemi on Vanda hybrids. Because of the popularity and high value of Vanda orchids, proper identification of this rapidly progressing bacterial disease is of great importance for the commercial producer and homeowner alike. References: (1) N. W. Schaad et al. Erwinia soft rot group. Page 56 in: Laboratory Guide for Identification of Plant Pathogenic Bacteria. 3rd ed. N. W. Schaad et al., eds. American Phytopathological Society. St. Paul, MN, 2001. (2) W. G. Weisburg. J. Bacteriol. 173:697, 1991.

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First Report of a Bacterial Soft Rot on Tolumnia Orchids Caused by a Dickeya sp. in the United States

Plant Disease ◽

10.1094/pdis-93-12-1354b ◽

2009 ◽

Vol 93 (12) ◽

pp. 1354-1354 ◽

Cited By ~ 8

Author(s):

R. A. Cating ◽

A. J. Palmateer ◽

R. T. McMillan ◽

E. R. Dickstein

Keyword(s):

Pathogenic Bacteria ◽

Soft Rot ◽

The United States ◽

South Florida ◽

Nutrient Agar ◽

Bacterial Suspension ◽

Brown Pigment ◽

Rrna Gene ◽

University Of Florida ◽

First Report

Tolumnia orchids are small epiphytic orchids grown for their attractive flowers. In the fall of 2008, approximately 100 Tolumnia orchids with soft, brown, macerated leaves were brought to the University of Florida Extension Plant Diagnostic Clinic in Homestead. Ten plants were randomly selected and bacteria were isolated from the margins of symptomatic tissues of each of the 10 plants on nutrient agar according to the method described by Schaad et al. (2). Four reference strains were used in all tests, including the molecular tests: Erwinia carotovora subsp. carotovora (obtained from J. Bartz, Department of Plant Pathology, University of Florida, Gainesville), E. chrysanthemi (ATCC No. 11662), Pectobacterium cypripedii (ATCC No. 29267), and Acidovorax avenae subsp. cattleyae (ATCC No. 10200). All 10 of the isolated bacteria were gram negative, grew at 37°C, degraded pectate in CVP (crystal violet pectate) medium, grew anaerobically, produced brown pigment on NGM (nutrient agar-glycerol-manganese chloride) medium (1), were sensitive to erythromycin, and produced phosphatase. Three of the strains were submitted for MIDI analysis (Sherlock version TSBA 4.10; Microbial Identification, Newark DE) (SIM 0.732 to 0.963), which identified them as E. chrysanthemi. A PCR assay was performed on the 16S rRNA gene with primers 27f and 1495r described by Weisburg et al. (3) from two of the isolates and a subsequent GenBank search showed 99% identity of the 1,508-bp sequence to that of Dickeya chrysanthemi (Accession No. FM946179) (formerly E. chrysanthemi). The sequences were deposited in GenBank (Accession Nos. GQ293897 and GQ293898). Pathogenicity was confirmed by injecting approximately 100 μl of a bacterial suspension at 1 × 108 CFU/ml into leaves of 10 Tolumnia orchid mericlones. Ten plants were also inoculated with water as controls. Plants were placed in a greenhouse at 29°C with 60 to 80% relative humidity. Within 24 h, soft rot symptoms appeared on all inoculated leaves. The water controls appeared normal. A Dickeya sp. was reisolated and identified using the above methods (biochemical tests and MIDI), fulfilling Koch's postulates. To our knowledge, this is the first report of a soft rot caused by a Dickeya sp. on Tolumnia orchids. Although 16S similarity and MIDI results suggest the isolated bacteria are D. chrysanthemi because of its close similarity with other Dickeya spp., these results are not conclusive. Further work should be conducted to confirm the identity of these isolates. Through correspondence with South Florida Tolumnia growers, it appears this disease has been a recurring problem, sometimes affecting international orchid shipments where plant losses have been in excess of 70%. References: (1) Y. A. Lee and C. P. Yu. J. Microbiol. Methods 64:200, 2006. (2) N. W. Schaad et al. Erwinia soft rot group. Page 56 in: Laboratory Guide for Identification of Plant Pathogenic Bacteria. 3rd ed. N. W. Schaad et al., eds. American Phytopathological Society. St. Paul, MN, 2001. (3) W. G. Weisburg et al. J. Bacteriol. 173:697, 1991.

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First Report of a Leaf Spot on Conyza sumatrensis Caused by Phoma macrostoma in China

Plant Disease ◽

10.1094/pdis-03-11-0228 ◽

2012 ◽

Vol 96 (1) ◽

pp. 148-148 ◽

Cited By ~ 1

Author(s):

J. Liu ◽

H. D. Luo ◽

W. Z. Tan ◽

L. Hu

Keyword(s):

Leaf Spot ◽

Fungal Pathogens ◽

Biological Diversity ◽

Biocontrol Agent ◽

Continuous Light ◽

The United States ◽

Leaf Spot Disease ◽

Pathogenicity Test ◽

Dry Land ◽

First Report

Conyza sumatrensis (Asteraceae), an annual or biennial plant, is native to North and South America. It is an invasive, noxious weed that is widespread in southern and southeastern China. It invades farm land and causes great losses to dry land crops, including wheat, corn, and beans. It also reduces biological diversity by crowding out native plants in the infested areas (3,4). During a search for fungal pathogens that could serve as potential biological control agents of C. sumatrensis, a leaf spot disease was observed in 2010 in Chongqing, China. An isolate (SMBC22) of a highly virulent fungus was obtained from diseased leaves. Pathogenicity tests were performed by placing 6-mm-diameter mycelial disks of 7-day-old potato dextrose agar (PDA) cultures of SMBC22 on leaves of 15 healthy greenhouse-grown plants of C. sumatrensis; the same number of control plants was treated with sterile PDA disks. Treated plants were covered with plastic bags for 24 h and maintained in a growth chamber with daily average temperatures of 24 to 26°C, continuous light (3,100 lux), and high relative humidity (>90%). Lesions similar to those observed in the field were first obvious on the SMBC22-inoculated leaves 3 days after inoculation. Symptoms became severe 7 to 9 days after inoculation. Control plants remained healthy. The fungus was reisolated from inoculated and diseased leaves and it was morphologically the same as SMBC22. The pathogenicity test was conducted three times. A survey of 10 southern and southeastern Chinese provinces revealed that the disease was widespread and it attacked leaves and stems of seedlings and mature plants of C. sumatrensis. Lesions on leaves were initially small, circular, and water soaked. The typical lesion was ovoid or fusiform, dark brown, and surrounded by a yellow halo. The spots coalesced to form large lesions and plants were often completely blighted. Fungal colonies of SMBC22 on PDA plates were initially white and turned dark gray. Colonies were circular with smooth edges with obvious rings of pycnidia on the surface. Aerial hyphae were short and dense. Pycnidia, black and immersed or semi-immersed in the medium, were visible after 12 days of incubation. Pycnidia were 72 to 140 μm in diameter. Conidia were produced in the pycnidia and were hyaline, unicellular, ellipsoidal, and 4.4 to 6.1 × 1.6 to 2.2 μm. To confirm identification of the fungus, genomic DNA was extracted from mycelia of a 7-day-old culture on PDA at 25°C (2). The internal transcribed spacer (ITS) gene of rDNA was amplified using primers ITS4/ITS5. The gene sequence was 524 bp long and registered in NCBI GenBank (No. HQ645974). BLAST analysis showed that the current sequence had 99% homology to an isolate of Phoma macrostoma (DQ 404792) from Cirsium arvense (Canada thistle) in Canada and reported to cause chlorotic symptoms on that host plant (1). To our knowledge, this is the first report of P. macrostoma causing disease on C. sumatrensis in China. P. macrostoma, thought of as a biocontrol agent of broadleaf weeds in Canada, has been patented in the United States. The current isolate of P. macrostoma is considered as a potential biocontrol agent of C. sumatrensis. References: (1) P. R. Graupner et al. J. Nat. Prod. 66:1558, 2004. (2) S. Takamatsu et al. Mycoscience 42:135, 2001. (3) W. Z. Tan et al. Page 177 in: Manual of Emergency Control Technology Invasive Pests in China. G. L. Zhang, ed. Science Press, Beijing, 2010. (4) C. Wang et al. J. Wuhan Bot. Res. 28:90, 2010.

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First Report of Leaf Spot of Rudbeckia hirta var. pulcherrima Caused by Septoria rudbeckiae in Korea

Plant Disease ◽

10.1094/pdis-02-12-0172-pdn ◽

2012 ◽

Vol 96 (6) ◽

pp. 911-911 ◽

Cited By ~ 1

Author(s):

J. H. Park ◽

S. E. Cho ◽

K. S. Han ◽

H. D. Shin

Keyword(s):

Leaf Spot ◽

The United States ◽

Flowering Plant ◽

Leaf Spot Disease ◽

Morphological Identification ◽

Its Sequence ◽

Conidial Suspension ◽

First Report ◽

Rudbeckia Hirta ◽

Close Phylogenetic Relationship

Rudbeckia hirta L. var. pulcherrima Farw. (synonym R. bicolor Nutt.), known as the black-eyed Susan, is a flowering plant belonging to the family Asteraceae. The plant is native to North America and was introduced to Korea for ornamental purposes in the 1950s. In July 2011, a previously unknown leaf spot was first observed on the plants in a public garden in Namyangju, Korea. Leaf spot symptoms developed from lower leaves as small, blackish brown lesions, which enlarged to 6 mm in diameter. In the later stages of disease development, each lesion was usually surrounded with a yellow halo, detracting from the beauty of the green leaves of the plant. A number of black pycnidia were present in diseased leaf tissue. Later, the disease was observed in several locations in Korea, including Pyeongchang, Hoengseong, and Yangpyeong. Voucher specimens were deposited at the Korea University Herbarium (KUS-F25894 and KUS-F26180). An isolate was obtained from KUS-F26180 and deposited at the Korean Agricultural Culture Collection (Accession No. KACC46694). Pycnidia were amphigenous, but mostly hypogenous, scattered, dark brown-to-rusty brown, globose, embedded in host tissue or partly erumpent, 50 to 80 μm in diameter, with ostioles 15 to 25 μm in diameter. Conidia were substraight to mildly curved, guttulate, hyaline, 25 to 50 × 1.5 to 2.5 μm, and one- to three-septate. Based on the morphological characteristics, the fungus was consistent with Septoria rudbeckiae Ellis & Halst. (1,3,4). Morphological identification of the fungus was confirmed by molecular data. Genomic DNA was extracted using the DNeasy Plant Mini DNA Extraction Kit (Qiagen Inc., Valencia, CA.). The internal transcribed spacer (ITS) region of rDNA was amplified using the ITS1/ITS4 primers and sequenced. The resulting sequence of 528 bp was deposited in GenBank (Accession No. JQ677043). A BLAST search showed that there was no matching sequence of S. rudbeckiae; therefore, this is the first ITS sequence of the species submitted to GenBank. The ITS sequence showed >99% similarity with those of many Septoria species, indicating their close phylogenetic relationship. Pathogenicity was tested by spraying leaves of three potted young plants with a conidial suspension (2 × 105 conidia/ml), which was harvested from a 4-week-old culture on potato dextrose agar. Control leaves were sprayed with sterile water. The plants were covered with plastic bags to maintain 100% relative humidity (RH) for the first 24 h. Plants were then maintained in a greenhouse (22 to 28°C and 70 to 80% RH). After 5 days, leaf spot symptoms identical to those observed in the field started to develop on the leaves inoculated with the fungus. No symptoms were observed on control plants. S. rudbeckiae was reisolated from the lesions of inoculated plants, confirming Koch's postulates. A leaf spot disease associated with S. rudbeckiae has been reported on several species of Rudbeckia in the United States, Romania, and Bulgaria (1–4). To our knowledge, this is the first report of leaf spot on R. hirta var. pulcherrima caused by S. rudbeckiae in Korea. References: (1) J. B. Ellis and B. D. Halsted. J. Mycol. 6:33, 1890. (2) D. F. Farr and A. Y. Rossman. Fungal Databases. Systematic Mycology and Microbiology Laboratory, ARS, USDA. Retrieved from http://nt.ars-grin.gov/fungaldatabases/ February 2, 2012. (3) E. Radulescu et al. Septoriozele din Romania. Ed. Acad. Rep. Soc. Romania, Bucuresti, Romania, 1973. (4) S. G. Vanev et al. Fungi Bulgaricae 3:1, 1997.

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First Report of Root-Knot Nematode Meloidogyne enterolobii on Poinsettia ‘Luv U Pink’ in Taiwan

Plant Disease ◽

10.1094/pdis-09-21-1899-pdn ◽

2021 ◽

Author(s):

Che-Chang Liang ◽

P. Janet Chen

Keyword(s):

United States ◽

18S Rrna ◽

The United States ◽

18S Rrna Gene ◽

Rrna Gene ◽

First Report ◽

Meloidogyne Enterolobii ◽

Mock Control ◽

Haplotype 1 ◽

Coii Region

Poinsettia (Euphorbia pulcherrima Willd. ex Klotzsch.), originated in southern Mexico and northern Guatemala, is the most valuable potted flowering plant in the spurge family (Euphorbiaceae). The European Union and the United States are two biggest poinsettia markets (Taylor et al. 2011), with a wholesale value of $153 million in the United States in 2019. Root knot galls of poinsettia ‘Luv U Pink’ were collected from a production greenhouse located in Nantou County, Taiwan in March 2021. No aboveground symptoms were observed. A nematode population was established from a single female and used for identification and the Koch’s postulate. The perineal patterns of randomly picked 5 females are round or ovoid with moderate to high dorsal arches, but no distinct lateral lines, ventral striae are fine and smooth. The Morphometric characters of second-stage juvenile include: a vermiform body shape, tail narrow and tapering with rounded tail tips, and a distinct hyaline tail end. Measurements of 20 J2 are as follows: body length, 430 (398 - 473) μm; body width, 15.4 (13.4 - 17.8) μm; stylet length,13.4 (13.0 - 14.0) μm; dorsal esophageal gland orifice to basal knob, 3.4 (2.8 - 3.9) μm; tail length, 52.9 (47.6 - 62.2) μm. All morphometric data were consistent with the original description of Meloidogyne enterolobii (Yang and Eisenback 1983). Nematode DNA was extracted using GeneMark Tissue & Cell Genomic DNA Purification Kit (GeneMark, Taiwan) from approximately 1500 J2 and used for amplification of 18S rRNA gene, a D2-D3 region of 28S rRNA gene, and a mtDNA COII region with primer sets 1A/MelR, D2A/D3B, and C2F3/1108, respectively (Power and Harris 1993, Subbotin et al. 2006, Tigano et al. 2005). The sequence of 18S rRNA gene (accession no. MZ948800 haplotype 1 and MZ955998 haplotype 2), haplotype 1 shared 100% identity with that of M. enterolobii from the United States (KP901058) and China (MN832688); haplotype 2 shared 99.8% identity with that of KP901058 and MN832688. The sequence of the D2-D3 region (MZ955995) shared 99% identity with that M. enterolobii from the United States (KP901079). Sequence of the COII region (MZ964625) also shared 99% identity with that of M. enterolobii from the United States (AY446975) and China (MN840970). Phylogenetic trees of the three gene sequences plotted as described by Ye et al. (2021) revealed that the newly described nematode was grouped with M. enterolobii. Sequence analysis of two fragments: 236 bp and 520 bp amplified with gene specific primers Me-F/R and MK7F/R, respectively (Long et al. 2006, Tigano et al. 2010) also confirmed the identity of M. enterolobii. To measure the reproductive factor (Rf), the Poinsettia ‘Luv U Pink’ seedlings with eight true leaves were transplanted into three 12-cm diameter pots each containing 6000 eggs or water (mock control). Forty-five days after inoculation, the average Rf value of three inoculated plants was 6, and no galls were observed on mock control plant roots, confirming that poinsettia is the host of M. enterolobii. M. enterolobii has been reported in several Euphorbia species, including E. heterophylla, E. prostrata, E. punicea and E. tirucalli (Han et al. 2012, Rich et al. 2009). To the best of our knowledge, this is the first report of M. enterolobii infecting E. pulcherrima ‘Luv U Pink’.

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First Report of Guignardia citricarpa Associated with Citrus Black Spot on Sweet Orange (Citrus sinensis) in North America

Plant Disease ◽

10.1094/pdis-01-12-0101-pdn ◽

2012 ◽

Vol 96 (8) ◽

pp. 1225-1225 ◽

Cited By ~ 31

Author(s):

T. S. Schubert ◽

M. M. Dewdney ◽

N. A. Peres ◽

M. E. Palm ◽

A. Jeyaprakash ◽

...

Keyword(s):

North America ◽

Sweet Orange ◽

Black Spot ◽

The United States ◽

Yield Reduction ◽

Rrna Gene ◽

Specific Primers ◽

Oatmeal Agar ◽

First Report ◽

Citrus Black Spot

In March 2010, citrus black spot symptoms were observed on sweet orange trees in a grove near Immokalee, FL. Symptoms observed on fruit included hard spot, cracked spot, and early virulent spot. Hard spot lesions were up to 5 mm, depressed with a chocolate margin and a necrotic, tan center, often with black pycnidia (140 to 200 μm) present. Cracked spot lesions were large (15 mm), dark brown, with diffuse margins and raised cracks. In some cases, hard spots formed in the center of lesions. Early virulent spot lesions were small (up to 7 mm long), bright red, irregular, indented, and often with many pycnidia. In addition, small (2 to 3 mm), elliptical, reddish brown leaf lesions with depressed tan centers were observed on some trees with symptomatic fruit. Chlorotic halos appeared as they aged. Most leaves had single lesions, occasionally up to four per leaf. Tissue pieces from hard spots and early virulent spots were placed aseptically on potato dextrose agar (PDA), oatmeal agar, or carrot agar and incubated with 12 h of light and dark at 24°C. Cultures that grew colonies within a week were discarded. Fourteen single-spore cultures were obtained from the isolates that grew slower than the Guignardia mangiferae reference cultures, although pycnidia formed more rapidly in the G. mangiferae cultures (1). No sexual structures were observed. Cultures on half-PDA were black and cordlike with irregular margins with numerous pycnidia, often bearing white cirrhi after 14 days. Conidia (7.1 to 7.8 × 10.3 to 11.8 μm) were hyaline, aseptate, multiguttulate, ovoid with a flattened base surrounded by a hyaline matrix (0.4 to 0.6 μm) and a hyaline appendage on the rounded apex, corresponding to published descriptions of G. citricarpa (anomorph Phyllosticta citricarpa) (1). A yellow pigment was seen in oatmeal agar surrounding G. citricarpa, but not G. mangiferae colonies as previously reported (1,2). DNA was extracted from lesions and cultures and amplified with species-specific primers (2). DNA was also extracted from G. mangiferae and healthy citrus fruit. The G. citricarpa-specific primers produced a 300-bp band from fruit lesions and pure cultures. G. mangiferae-specific primers produced 290-bp bands with DNA from G. mangiferae cultures. The internally transcribed spacer (ITS) of the rRNA gene, translation-elongation factor (TEF), and actin gene regions were sequenced from G. citricarpa isolates and deposited in GenBank. These sequences had 100% homology with G. citricarpa ITS sequences from South Africa and Brazil, 100% homology with TEF, and 99% homology with actin of a Brazilian isolate. Pathogenicity tests with G. citricarpa were not done because the organism infects immature fruit and has an incubation period of at least 6 months (3). In addition, quarantine restrictions limit work with the organism outside a contained facility. To our knowledge, this is the first report of black spot in North America. The initial infested area was ~57 km2. The disease is of great importance to the Florida citrus industry because it causes serious blemishes and significant yield reduction, especially on the most commonly grown ‘Valencia’ sweet orange. Also, the presence of the disease in Florida may affect market access because G. citricarpa is considered a quarantine pathogen by the United States and internationally. References: (1) R. P. Baayen et al. Phytopathology 92:464, 2002. (2) N. A. Peres et al. Plant Dis. 91:525, 2007 (3) R. F. Reis et al. Fitopath Bras. 31:29, 2006.

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Isolation and Identification of the Causal Agent of Brown Stalk Rot, A New Disease of Maize in South Africa

Plant Disease ◽

10.1094/pdis-91-6-0711 ◽

2007 ◽

Vol 91 (6) ◽

pp. 711-718 ◽

Cited By ~ 26

Author(s):

T. Goszczynska ◽

W. J. Botha ◽

S. N. Venter ◽

T. A. Coutinho

Keyword(s):

South Africa ◽

Biochemical Characterization ◽

Bacterial Species ◽

Anaerobic Bacteria ◽

Rrna Gene ◽

New Disease ◽

Isolation And Identification ◽

Stalk Rot ◽

Pathogenicity Tests ◽

Internal Browning

During 2004 to 2005, an unreported disease of maize (Zea mays) was observed on commercial fields in the Northwest and Mpumalanga Provinces of South Africa. Infected plants were stunted, with a vertical crack at the first internode. Inside the stem, a dark-brown, narrow lesion was present along the crack. Internal browning inside the stem extended upward, reaching the top internode in some plants. Seed cobs were underdeveloped. Diseased plants were scattered in the fields and 10 to 70% of the crop was affected. Gram-negative, facultatively anaerobic bacteria were consistently isolated from diseased tissues. Pathogenicity tests established that representative strains induced disease symptoms similar to those observed on maize plants in the field. Physiological and biochemical characterization using the API 20E and API 50CHE systems and 16S rRNA gene sequence analyses showed that the strains belonged to the genus Pantoea. The results of these tests also separated the strains into two groups. The first group, giving a positive reaction in the indole test, was similar to Pantoea ananatis. The second group of strains was indole negative and resembled P. agglomerans. The fluorescent amplified fragment length polymorphism (F-AFLP) genomic fingerprints generated by the indole-positive strains and P. ananatis reference strains were similar and clustered together in the dendrogram, confirming that the indole-positive bacteria causing brown stalk rot on maize were P. ananatis. The F-AFLP fingerprints produced by the indole-negative strains were distinctly different from those generated by P. ananatis, P. agglomerans, P. dispersa, P. citrea, P. stewartii subsp. stewartii, and P. stewartii subsp. indologenes. The results indicated that indole-negative bacteria causing brown stalk rot on maize might belong to a previously undescribed species of the genus Pantoea. This is the first report of a new disease on maize, brown stalk rot, caused by two bacterial species, P. ananatis and an undescribed Pantoea sp.

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Secondary infection by anaerobic bacteria possibly ensues a battle for oxygen in SARS-Cov2 infected patients: anaerobe-targeting antibiotics (like doxycycline/Metronidazole) to supplement Azithromycin in the treatment regimen of COVID19?

10.31219/osf.io/s48fv ◽

2020 ◽

Author(s):

Sandeep Chakraborty ◽

Gautam Das

Keyword(s):

Health Care ◽

Treatment Regimen ◽

Health Care Providers ◽

Pathogenic Bacteria ◽

Bacterial Species ◽

Anaerobic Bacteria ◽

Care Providers ◽

Rna Seq ◽

Sequencing Data ◽

Hemoglobin Degradation

The Covid19 pandemic [1], triggered by novel strain of a coronavirus SARS-Cov2 [2] first detected in Wuhan City, China, has spread globally like a wildfire [3], resulting in significant loss of life [4] and endangering health care providers and community health care workers [5]. Understanding and interpreting the underlying metagenome of this disease will help provide direction for the right treatment regimen.RNA-sequencing as a more sensitive and comprehensive diagnostic test:RNA-sequencing is a more sensitive and comprehensive test (albeit more time-consuming and expensive), providing information on a larger range of organisms (metagenomic profile) present in the patient sample in comparison to reverse transcription PCR or antibody-testing. For example in one study, Covid19 patients were tested for four bacterial species, including Mycoplasma, with negative results. However, the sequencing data clearly reveals the presence of Mycoplasma [6]. Another study of 2 patients in Wuhan [7] used the Metaphlan2 program to conclude that Capnocytophaga and Veillonella are the only bacterial species present in one patient, and found none in another, when this clearly was not the case [8].Other RNA-sequencing data submitted in NCBI has identified several potentially pathogenic bacteria in multiple patient samples from across the globe [6,9–12]. The obligate anaerobe Prevotella had signifi- cant abundance in one patient, over-expressing immune-suppression proteins [13]. While all studies reveal Prevotella in varying abundance, other bacteria (Lautropia, Cutibacterium, Haemophilus, Pseudomonas, Sphingomonas etc.) are also found in significant quantity to attract attention to secondary infections [6,12]. RNA-seq also reveals extremely low viral loads in many patients, explaining the high false negatives (8 times negative before a positive) [14] and failure to detect virus using RT-PCR in severely sick patients, who were CT+ve [15].Anaerobic bacteria hypothesis:Recent studies from Italy have suggested that Covid-19 does not lead to a ‘“typical” acute respiratory distress syndrome (ARDS)’ [16]. Furthermore, elevated D-dimer levels suggest hemoglobin degradation leading to coagulation [17,18].A simple hypothesis that emerges from RNA-seq data is over-representation of anaerobic bacteria in Covid19 patients, not found in BALF samples from normal patients (unpublished data), in a battle for oxygen. These bacteria express hemoglobin degrading proteins [19], heme-binding proteins sequestering heme after hemoglobin degradation [20], ‘plundering‘ iron, and thereby sequestering oxygen [21]. Hypoxia could also result from formate, the by-product of anaerobic respiration, which inhibits mitochondrial cytochrome oxidase, causing hypoxia at the cellular level [22].Our proposal for anaerobe-specific antibiotics as a therapy:We propose the use of anaerobe-specific antibiotics, like Metronidazole, in the treatment regimen to supple- ment the successfully used doxycycline/Azithromycin antibiotic [23], along with anti-coagulants [24].

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First Report of Leaf Spot Disease On Microstegium vimineum Caused by Bipolaris setariae in China

Plant Disease ◽

10.1094/pdis-04-21-0703-pdn ◽

2021 ◽

Author(s):

Min Tan ◽

Qiong Huang ◽

Hao Fan ◽

Yun Wu ◽

Richard C. Reardon ◽

...

Keyword(s):

United States ◽

Leaf Spot ◽

The United States ◽

Lethal Concentration ◽

Reference Sequence ◽

Microstegium Vimineum ◽

Morphological Observation ◽

Leaf Spot Disease ◽

First Report ◽

Spot Disease

Microstegium vimineum, a Poaceae annual C4 plant, occurred widely in crop fields, tea gardens, orchards, under forests and roadsides in most provinces and regions south of the Yellow River, China. It was introduced into the eastern USA causing ecological and environmental damage (Stricker, 2016). In October 2015, M. vimineum plants with leaf spots were observed on the roadside of Mingling Road (32.04521°E, 118.84323°N), Nanjing, China. In an early stage of disease development, light brown or brown, round or oval shaped lesions appeared on the upper surface of leaves. In a middle stage, the lesions gradually expanded and the edges of the diseased leaves were lightly curled. In a late stage, leaves were withered or curled and the entire plant died. Initial disease incidence was up to 85% among natural populations of the weed. Diseased leaves collected from field were surface disinfected (75% ethanol for 30s; 1% sodium hypochlorite solution for 30s; 75% ethanol for 30s; sterile deionized water for 1min) and placed on water agar (20g agar per liter) (Kleczewski et al., 2010). Plates were incubated in the dark at 28℃ for 3 days. Following incubation, leaves, spores and conidiophores were examined using light microscopy. Single spores were obtained by using the single-spore procedure, plating out a loopful of spores onto water agar, and then carving individual spores out with associated agar under a microscope. Single spores were isolated, plated onto MV-agar (30g M. vimineum leaves, 20g agar per liter), and placed under 365 nm wavelength black light. Fungal colonies were transferred onto PDA medium, after 4 days colonies measured between 83 to 86 mm in diameter, appeared flat and dark brown, with short, light gray aerial hyphae. Conidiophores were solitary or clustered, light brown to medium brown, with pale apical color and multiple septa. The upper part was usually geniculated, 5.5-9.5 μm wide. Conidia were light yellowish brown to medium yellowish brown, mostly fusiform, straight or curved, fusoid or navicular, often slightly curved, rarely straight, smooth, 5-9 (mostly 7) septa, 48-70×10-14.5 μm (average 57×12.5 μm); hilium slightly prominent, and truncated at the base. Through morphological observation, the fungus was preliminarily identified as Bipolaris sp.. Four to five seeds of M. vimineum were planted in pots (10 cm in diameter) filled with nutrient soil, placed in the greenhouse and watered regularly. Four pots were inoculated with a conidia suspension of 1×105 sp/mL, at 4-5 true stage. Inoculated seedlings were maintained under 80% humidity and 28℃ for 24h in the dark, and then transferred to a greenhouse. Three pots of uninoculated seedlings were used as controls. Two days after the inoculation, buff-colored, irregular-shaped spots appeared centered on leaf veins. Within a week, diseased leaves became crinkled and their edges were yellow to brown due to proliferation of the spots. By 15 days, large areas of brown spots appeared on the leaves, some leaves turned yellow-brown and severely curled, and 80% of the plants had died. The diseased symptoms were similar to that of the field sample. The fungus re-isolated resulted morphologically identical to the original isolate grown on PDA medium and used for inoculation, thus fulfilled Koch’s postulates. The CTAB method was used to extract DNA from isolates of diseased leaves taken directly from the field, and the internal transcribed spacer (ITS) and glyceraldehyde 3-phosphate dehydrogenase gene (GPDH) were amplified using primer pairs of ITS1/ITS4 and GPD/GPD2 (Manamgoda et al., 2014) respectively. The ITS amplified sequence (Genbank accession MW446193) shared 100% identity with the reference sequence of Bipolaris setariae (MN215638.1) and the GPDH amplified sequence (MW464364) shared 99.83% identity with the reference sequence of B. setariae (MK144540.1). Field experiments were conducted in Laboratory Base of Nanjing Agricultural University, where M. vimineum plants were planted. Spore suspensions with concentrations of 105, 104, 103, 102, and 101 sp/mL were prepared, distilled water was used for control, and there were four replicates of each treatment. Twenty four plots were randomly arranged, the experimental unit consisted of 50 to 60 plants in an area of 0.5m×0.6m. The interval distance between plots was about 20 cm so as to prevent the mutual influence among treatments. M. vimineum plants were inoculated at 3-4 true leaf stage. Inoculation was done at sunset, and 60 mL spore suspension was sprayed onto each plot. After spraying, the waterproof-breathable black cloth was used to cover the plots, and removed 36 hours later. The outdoor temperature was 20~28℃. After 10 days, the symptoms of M. vimineum were observed and the disease index was recorded. SPSS 20 software (SPSS Inc., Chicago, IL, USA) was used for variance analysis, and Origin 9.0 (OriginLab, Hampton, MA, USA) was used to calculate the half lethal concentration (ED50) and 90% lethal concentration (ED90) of the strain MLL-1-5 on M. vimineum. Symptoms appeared on inoculated M. vimineum seedlings immediately after dark treatment. Within a week, all seedlings inoculated with the highest spore concentration were dead. Plants sprayed with water remained healthy. ED50 and ED90 of the strain MLL-1-5 was 1.9×101 and 1.4×103 sp/mL respectively, which indicated aggressiveness of the strain MLL-1-5 B. setariae. After 28 days, infected M. vimineum plants did not recover. This is the first report of leaf spot disease on M. vimineum caused by B. setariae in China. M. vimineum is a widely distributed and extremely harmful weed in China and United States. No biocontrol agents against M. vimineum are currently available. B. setariae may have potential as a biocontrol agent against M. vimineum both in China and the United States.

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First Report of New Bacterial Leaf Blight of Rice Caused by Pantoea ananatis in Southeast China

Plant Disease ◽

10.1094/pdis-05-21-0988-pdn ◽

2021 ◽

Author(s):

Lin Yu ◽

Changdeng Yang ◽

Zhijuan Ji ◽

Yuxiang Zeng ◽

Yan Liang ◽

...

Keyword(s):

Oryza Sativa L ◽

Sequence Similarity ◽

Disease Incidence ◽

Leaf Blight ◽

Bacterial Leaf Blight ◽

Bacterial Suspension ◽

Rrna Gene ◽

Pantoea Ananatis ◽

Southeast China ◽

First Report

In autumn 2020, leaf blight was observed on rice (Oryza sativa L., variety Zhongzao39, Yongyou9, Yongyou12, Yongyou15, Yongyou18, Yongyou1540, Zhongzheyou8, Jiafengyou2, Xiangliangyou900 and Jiyou351) in the fields of 17 towns in Zhejiang and Jiangxi Provinces, China. The disease incidence was 45%-60%. Initially, water-soaked, linear, light brown lesions emerged in the upper blades of the leaves, and then spread down to leaf margins, which ultimately caused leaf curling and blight during the booting-harvest stage (Fig. S1). The disease symptoms were assumed to be caused by Xanthomonas oryzae pv. oryzae (Xoo), the pathogen of rice bacterial blight. 63 isolates were obtained from the collected diseased leaves as previously described (Hou et al. 2020). All isolates showed circular, smooth-margined, yellow colonies when cultured on peptone sugar agar (PSA) medium for 24h at 28℃. The cells were all gram-negative and rod-shaped with three to six peritrichous flagella; positive for catalase, indole, glucose fermentation and citrate utilization, while negative for oxidase, alkaline, phenylalanine deaminase, urease, and nitrate reductase reactions. 16S rRNA gene sequence analysis from the 6 isolates (FY43, JH31, JH99, TZ20, TZ39 and TZ68) revealed that the amplified fragments shared 98% similarity with Pantoea ananatis type strain LMG 2665T (GenBank JFZU01) (Table S3). To further verify P. ananatis identity of these isolates, fragments of three housekeeping genes including gyrB, leuS and rpoB from the 6 isolates were amplified and sequenced, which showed highest homology to LMG 2665T with a sequence similarity of 95%-100% (Table S3). Primers (Brady et al. 2008) and GenBank accession numbers of gene sequences from the 6 isolates are listed in Table S1 and Table S2. Phylogenetic analysis of gyrB, leuS and rpoB concatenated sequences indicated that the 6 isolates were clustered in a stable branch with P. ananatis (Fig. S2). Based on the above morphological, physiological, biochemical and molecular data, the isolates are identified as P. ananatis. For pathogenicity tests, bacterial suspension at 108 CFU/mL was inoculated into flag leaves of rice (cv. Zhongzao39) at the late booting stage using clipping method. Water was used as a negative control. The clipped leaves displayed water-soaked lesions at 3 to 5 days after inoculation (DAI); then the lesion spread downward and turned light brown. At about 14 DAI, blight was shown with similar symptoms to those samples collected from the rice field of Zhejiang and Jiangxi provinces (Fig. S1). In contrast, the control plants remained healthy and symptomless. The same P. ananatis was re-isolated in the inoculated rice plants, fulfilling Koch’s postulates. In the past decade, P. ananatis has been reported to cause grain discoloration in Hangzhou, China (Yan et al. 2010) and induce leaf blight as a companion of Enterobacter asburiae in Sichuan province, China (Xue et al. 2020). Nevertheless, to the best of our knowledge, this is the first report of P. ananatis as the causative agent of rice leaf blight in southeast China. This study raises the alarm that the emerging rice bacterial leaf blight in southeast China might be caused by a new pathogen P. ananatis, instead of Xoo as traditionally assumed. Further, the differences of occurrence, spread and control between two rice bacterial leaf blight diseases caused by P. ananatis and Xoo, respectively need to be determined in the future.

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