First Report of Witches'-Broom Disease of Chinese Wingnut in China and its Association with a Phytoplasma of Aster Yellows Group (16SrI)

Pterocarya stenoptera C. DC., commonly known as Chinese wingnut, is a fast-growing deciduous tree with tough bark and attractive foliage. Because of its tolerance of compact and nutritionally poor soil, drought, and heat, Chinese wingnut is an important component of the biological diversity in natural ecosystems and is a favorable shade tree in China. Chinese wingnut has also been used as a rootstock for walnuts because of its high resistance to soilborne Phytophthora spp. In the spring of 2004, a disease characterized by witches'-broom symptoms was observed affecting Chinese wingnut trees growing in suburban Taian, Shandong, China. The diseased trees developed dense clusters of highly proliferating branches with shortened internodes, leaves on the affected branches were significantly smaller, and some branches and twigs suffered dieback. Phytoplasma infection was suspected as the cause of this Chinese wingnut witches'-broom (CWWB) disease because the disease occurred in an area where phytoplasmal diseases, such as paulownia witches'-broom (PaWB) and jujube witches'-broom (JWB), are common (3). Nested polymerase chain reactions (PCR) were performed on DNA samples extracted from leaves of six diseased trees using phytoplasma-universal 16S rDNA primers (R16mF2/R16mR1 and R16F2n/ R16R2) (1,2). Results revealed that all diseased trees examined were infected by phytoplasma, whereas PCR assays of leaf samples from two nearby symptomless Chinese wingnut trees were negative. Subsequent restriction fragment length polymorphism analysis of the PCR-amplified 16S rDNA indicated that all diseased trees contained the same phytoplasma and that the CWWB phytoplasma belongs to subgroup B of the “Candidatus Phytoplasma asteris” (AY) group (16SrI). Nucleotide sequence analysis of a 16S rRNA gene cloned from CWWB phytoplasma (GenBank Accession No. AY831966) suggested that this phytoplasma is closely related to, but distinct from, PaWB phytoplasma, another member of group16SrI. To our knowledge, this is the first report of Chinese wingnut witches'-broom disease and of its association with a phytoplasma. Further work is being undertaken to examine the ecological and evolutionary relationship between CWWB phytoplasma and other phytoplasmas in the region and to assess the impact of CWWB on walnut rootstock selection. References: (1) D. E. Gundersen and I.-M. Lee. Phytopathol. Mediterr. 35:144, 1996. (2) C. D. Smart et al. Appl. Environ. Microbiol. 62:2988, 1996. (3) S. Zhu et al. Acta Hortic. 472:701, 1998.

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First Report of Witches'-Broom Disease of Broussonetia papyrifera and Its Association with a Phytoplasma of Elm Yellows Group (16SrV)

Plant Disease ◽

10.1094/pdis.2004.88.7.770c ◽

2004 ◽

Vol 88 (7) ◽

pp. 770-770 ◽

Cited By ~ 8

Author(s):

Qingzhong Liu ◽

Tianqi Wu ◽

Robert E. Davis ◽

Yan Zhao

Keyword(s):

16S Rdna ◽

Biological Diversity ◽

23S Rrna ◽

Nucleotide Sequence Analysis ◽

Rrna Gene ◽

Natural Ecosystems ◽

Broussonetia Papyrifera ◽

First Report ◽

Broom Disease ◽

Elm Yellows

Broussonetia papyrifera, commonly known as paper mulberry, is an ornamental tree that is native to northeastern Asia. Because of its fast-growing nature and tolerance of dust, smoke, and high temperatures, paper mulberry is an important component of the biological diversity in natural ecosystems as well as a favorable shade tree in the region. In September of 2003, a disease characterized by pronounced witches'-broom symptoms was observed in paper mulberry trees growing near a jujube (Ziziphus jujuba) orchard and in home gardens located in Taian, Shandong, China. The diseased trees developed dense clusters of highly proliferating branches with shortened internodes. Leaves on the affected branches were chlorotic and greatly reduced in size. Phytoplasma infection was first suspected in this paper mulberry witches'-broom (PMWB) disease because the disease occurred in an area where other phytoplasmal diseases, including jujube witches'-broom (JWB) disease and paulownia witches'-broom (PaWB) disease, are common (4). Results from nested polymerase chain reactions (PCR), performed using phytoplasma-universal 16S rDNA primers (P1/P7 and R16F2n/R16R2) (1,2,3), revealed that all seven diseased trees tested contained phytoplasma, whereas PCR assay of comparable leaf samples from three nearby symptomless paper mulberry trees were negative. Subsequent restriction fragment length polymorphism (RFLP) analysis of the PCR-amplified 16S rDNA indicated that all diseased trees contained the same phytoplasma and that the PMWB phytoplasma belongs to the subgroup B of the elm yellows (EY) phytoplasma group (16SrV-B). Nucleotide sequence analysis of the cloned PMWB phytoplasma partial rRNA operon (GenBank Accession No. AY576685), spanning a near full-length 16S rRNA gene, a 16S–23S rRNA intergenic spacer, a tRNA-Ile gene, and a partial 23S rRNA gene, suggested that PMWB phytoplasma is most closely related to JWB phytoplasma, a member of the subgroup16SrV-B. To our knowledge, this is the first report of a paper mulberry witches'-broom disease and the first report of its association with a phytoplasma. Further work is underway to determine whether the PMWB phytoplasma is distinct from previously characterized phytoplasmas included in group 16SrV and to assess impacts of the phytoplasma on the ecosystems in the region. References: (1) S. Deng and C. Hiruki. J. Microbiol. Methods 14:53, 1991. (2) D. E. Gundersen and I.-M. Lee. Phytopathol. Mediterr. 35:144, 1996. (3) C. D. Smart et al. Appl. Environ. Microbiol. 62:2988, 1996. (4) S. Zhu et al. Acta Hortic. 472:701, 1998.

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First Report of a Natural Infection of Opuntia sp. by a ‘Candidatus Phytoplasma asteris’-Related Phytoplasma in China

Plant Disease ◽

10.1094/pdis-91-4-0461c ◽

2007 ◽

Vol 91 (4) ◽

pp. 461-461 ◽

Cited By ~ 3

Author(s):

W. Wei ◽

H. Cai ◽

H. Chen ◽

R. E. Davis ◽

Y. Zhao

Keyword(s):

16S Rdna ◽

Natural Infection ◽

Geographical Location ◽

Rflp Analysis ◽

23S Rrna ◽

Nucleotide Sequence Analysis ◽

Rrna Gene ◽

First Report ◽

Aesthetic Appearance ◽

Geographical Regions

Cacti (Opuntia spp.) are perennial, evergreen, succulent plants native to arid areas of the Americas. Because of their aesthetic appearance, many cacti have been cultivated and introduced to other parts of the world as ornamentals. Cacti are susceptible to phytoplasma infections and develop witches'-broom (WB) disease. Currently, all reported cactus WB cases are associated with infections by phytoplasmas in the peanut witches'-broom group (16SrII) (1,2,4). During a phytoplasma diversity survey carried out during 2004 in Yunnan, China, we collected 29 malformed and 14 healthy-looking naturally occurring cactus plants from 14 locations representing five geographical regions. Each of the 29 malformed plants exhibited stunted growth and possessed clusters of highly proliferating cladodia, typical symptoms of cactus WB disease. Nested-PCR was carried out on the DNA samples extracted from young cladodia of these plants using phytoplasma-universal 16S rDNA primers P1A/P7A and R16F2n/R16R2 (3). Results revealed that all 29 diseased plants that were examined were infected by phytoplasmas, whereas all 14 healthy-looking plants were negative for phytoplasmas. Subsequent restriction fragment length polymorphism (RFLP) analysis of the PCR-amplified 1.25-kb 16S rDNA fragments indicated that 28 diseased plants were infected by a phytoplasma of group 16SrII, whereas one plant (from Suan Village) was infected by a ‘Candidatus Phytoplasma asteris’-related (group 16SrI) phytoplasma designated as strain YN26. Nucleotide sequence analysis of the strain YN26 partial rRNA operon (GenBank Accession No. EF190970), covering a near full-length 16S rRNA gene, a 16S-23S rRNA intergenic spacer, a tRNA-Ile gene, and a partial 23S rRNA gene, suggested that this phytoplasma is most closely related to an ash witches'-broom phytoplasma (GenBank Accession No. AY566302, 99.7% identity) and an epilobium phyllody phytoplasma (GenBank Accession No. AY101386, 99.7% identity), both members of subgroup16SrI-B. This YN26-infected cactus plant was transferred to a greenhouse and maintained for more than 2 years, during which time DNA samples were extracted and tested two additional times. The same 16S rDNA RFLP pattern type was consistently obtained in these tests, confirming that the plant remained infected by the 16SrI phytoplasma. To our knowledge, this is the first report of a natural infection of a cactus species by a group 16SrI phytoplasma. Since this 16SrI-cactus WB phytoplasma was found in the same geographical location where 16SrII-cactus WB phytoplasma was detected both in this and a previous study (1), the findings raised the question whether 16SrI- and 16SrII-cactus WB phytoplasmas have overlapping geo- and bioecological niches. References: (1) H. Cai et al. Plant Pathol. 51:394, 2002. (2) E. Choueiri et al. Plant Dis. 89:1129, 2005. (3) I. M. Lee et al. Int. J. Syst. Evol. Microbiol 54:337, 2004. (4) N. Leyva-Lopez et al. Phytopathology (Abstr.) 89(suppl):S45, 1999.

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First Report of Witches'-Broom Disease in a Cannabis spp. in China and Its Association with a Phytoplasma of Elm Yellows Group (16SrV)

Plant Disease ◽

10.1094/pdis-91-2-0227c ◽

2007 ◽

Vol 91 (2) ◽

pp. 227-227 ◽

Cited By ~ 11

Author(s):

Y. Zhao ◽

Q. Sun ◽

R. E. Davis ◽

I.-M. Lee ◽

Q. Liu

Keyword(s):

Nucleotide Sequence ◽

16S Rdna ◽

Geographic Location ◽

Full Length ◽

23S Rrna ◽

Nucleotide Sequence Analysis ◽

Rrna Gene ◽

Hemp Fiber ◽

First Report ◽

Elm Yellows

Hemp fiber plants (Cannabis spp.) spread naturally in almost every climate zone in China and have a long history of cultivation in the country (1). While hemp stalks provide high-quality fibers for making ropes, clothes, and paper products, hemp seeds are a rich source of edible oil. During the summer of 2004, a disease characterized by witches'-broom symptoms was observed in wild hemp fiber plants growing in suburban Taian, Shandong, China. The diseased plants developed clusters of highly proliferating branches with much shortened internodes and leaves on the affected branches were significantly reduced in size. Phytoplasma infection was suspected in this hemp fiber witches'-broom (HFWB) disease because of the typical symptoms and because of its geographic location where other phytoplasmal diseases such as jujube witches'-broom (JWB), paulownia witches'-broom (PaWB), paper mulberry witches'-broom (PMWB), and Chinese wingnut witches'-broom (CWWB) diseases were previously reported (3,4). Total DNA was extracted from leaves of four diseased and four nearby healthy looking hemp fiber plants. Nested PCR were carried out on the DNA samples using phytoplasma universal 16S rDNA primers (P1A/16S-SR and R16F2n/R16R2) (2). Results revealed that all examined diseased plants were infected by phytoplasma, whereas nearby healthy looking plants were phytoplasma free. Subsequent restriction fragment length polymorphism (RFLP) analysis of the PCR-amplified 1.25-kb 16S rDNA R16F2n/R16R2 fragment indicated that the phytoplasma associated with HFWB disease belongs to subgroup 16SrV-B of the elm yellows (EY) phytoplasma group. Nucleotide sequence analysis of the cloned HFWB phytoplasma partial rRNA operon (GenBank Accession No. EF029092), spanning a near full-length 16S rRNA gene and a partial 16S-23S rRNA intergenic spacer, suggested that HFWB phytoplasma is most closely related to JWB and PMWB phytoplasmas, both members of subgroup16SrV-B. To further characterize the HFWB phytoplasma, a genomic segment covering full-length ribosomal protein genes rplV and rpsC was PCR-amplified using primer pair rp(V)F1A/rp(V)R1A (2), cloned, and sequenced (GenBank Accession No. EF029093). The nucleotide sequence of the HFWB phytoplasma rplV and rpsC locus is nearly identical (99.9%) to that of JWB phytoplasma. To our knowledge, this is the first report of a phytoplasmal disease in Cannabis spp. Since HFWB and JWB phytoplasmas share extremely high sequence identity and share the same eco-geographic location, further investigation is warranted to determine whether these two phytoplasmas are actually one species that can infect both plants, an issue having important implications in managing both diseases. References: (1) S. Hong and R. C. Clarke. J. Int. Hemp Assoc. 3:55, 1996. (2) I. M. Lee et al. Int. J. Syst. Evol. Microbiol. 54:337, 2004. (3) Q. Liu et al. Plant Dis. 88:770, 2004. (4) Q. Liu et al. Plant Dis. 89:529, 2005.

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First Report of a Group 16SrII Phytoplasma Associated with Witches'-Broom of Eggplant in Oman

Plant Disease ◽

10.1094/pdis-10-10-0761 ◽

2011 ◽

Vol 95 (3) ◽

pp. 360-360 ◽

Cited By ~ 9

Author(s):

A. M. Al-Subhi ◽

N. A. Al-Saady ◽

A. J. Khan ◽

M. L. Deadman

Keyword(s):

Nested Pcr ◽

Solanum Melongena ◽

Positive Control ◽

Rrna Gene ◽

Direct Pcr ◽

First Report ◽

Pcr Product ◽

Pcr Products ◽

Broom Disease ◽

Phytoplasma Infection

Eggplant (Solanum melongena L.) belongs to the family Solanaceae and is an important vegetable cash crop grown in most parts of Oman. In February 2010, plants showing phyllody symptoms and proliferation of shoots resembling those caused by phytoplasma infection were observed at Khasab, 500 km north of Muscat. Total genomic DNA was extracted from healthy and two symptomatic plants with a modified (CTAB) buffer method (2) and analyzed by direct and nested PCR with universal phytoplasma 16S rDNA primers P1/P7 and R16F2n/ R16R2, respectively. PCR amplifications from all infected plants yielded an expected product of 1.8 kb with P1/P7 primers and a 1.2-kb fragment with nested PCR, while no products were evident with DNA from healthy plants. Restriction fragment length polymorphism (RFLP) profiles of the 1.2-kb nested PCR products of two eggplant phyllody phytoplasma and five phytoplasma control strains belonging to different groups used as positive control were generated with the restriction endonucleases RsaI, AluI, Tru9I, T-HB8I, and HpaII. The eggplant phytoplasma DNA yielded patterns similar to alfalfa witches'-broom phytoplasma (GenBank Accession No. AF438413) belonging to subgroup 16SrII-D, which has been recorded in Oman (1). The DNA sequence of the 1.8-kb direct PCR product was deposited in GenBank (Accession No. HQ423156). Sequence homology results using BLAST revealed that the eggplant phyllody phytoplasma shared >99% sequence identity with Scaevola witches'-broom phytoplasma (Accession No. AB257291.1), eggplant phyllody phytoplasma (Accession No. FN257482.1), and alfalfa witches'-broom phytoplasma (Accession No. AY169323). The RFLP and BLAST results of 16S rRNA gene sequences confirm that eggplant phyllody phytoplasma is similar to the alfalfa phytoplasma belonging to subgroup 16SrII-D. To our knowledge, this is the first report of a phytoplasma of the 16SrII-D group causing witches'-broom disease on eggplant in Oman. References: (1) A. J. Khan et al. Phytopathology 92:1038, 2002. (2) M. A. Saghai-Maroof et al. Proc. Natl. Acad. Sci. USA, 81:8014, 1984.

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First Report of a 16SrI-C Group Phytoplasma Associated With a Yellows-Type Disease Affecting Willow Plants in China

Plant Disease ◽

10.1094/pdis-93-2-0197b ◽

2009 ◽

Vol 93 (2) ◽

pp. 197-197 ◽

Cited By ~ 4

Author(s):

T. Wei ◽

Y. F. Wu ◽

K. K. Wu ◽

W. Hou ◽

Y. R. Li

Keyword(s):

16S Rdna ◽

Rflp Analysis ◽

Northern China ◽

Shaanxi Province ◽

Nucleotide Sequence Analysis ◽

Molecular Evidence ◽

Rrna Gene ◽

Transmission Electron ◽

Ctab Method ◽

Salix Babylonica

In May of 2008, a phytoplasma-like disease was observed on willows (Salix babylonica Linn) grown in the Shaanxi Province. Affected plants showed yellowed leaves with green veins and dieback. Incidence of the disease was less than 10%. Samples were collected from 10 symptomatic and five asymptomatic willow plants from five different areas in Shaanxi Province. Total DNA was extracted from 0.5 g of leaf midrib and stem phloem tissue with a modified cetyltrimethylammoniumbromide (CTAB) method (3). Resulting DNA extracts were analyzed by a nested PCR assay using phytoplasma 16S rRNA gene primer pairs R16mF2/R16mR1 followed by R16F2n/R16R2 (1), which amplified a 1,452- and a 1,246-bp product, respectively. Sequences of amplicons were almost the same. Restriction fragment length polymorphism (RFLP) analysis of the nested 1.2-kb 16S rDNA products with AluI, MseI, HhaI, HpaI, RsaI, HinfI, and TaqI endonucleases (2) indicated that all symptomatic plants were infected by a phytoplasma belonging to aster yellows group (16SrI) subgroup C (16SrI-C) ‘Candidatus Phytoplasma asteris’. None of the symptomless plants tested positive. Nucleotide sequence analysis of cloned 16S rDNA (GenBank Accession No. FJ179166) confirmed the results on the basis of RFLP analyses. Subsequently, the presence of the phytoplasmas in symptomatic plants was also confirmed by transmission electron microscopy. To our knowledge, this is the first molecular evidence of the presence of a phytoplasma associated with a yellows-type disease of willows in northern China and its association with aster yellow group 16SrI, subgroup 16SrI-C. References: (1) D. E. Gundersen and I.-M. Lee. Phytopathol. Mediterr. 35:144, 1996. (2) I.-M. Lee et al. Inst. J. Syst. Bacteriol. 48:1153, 1998. (3) Y. Qi et al. Biotechnol. Bull. 4:44, 2004.

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Characterization of halophilic bacteria from environmental samples from the brackish water of Pulicat Lake, India

Biologia ◽

10.2478/s11756-011-0094-2 ◽

2011 ◽

Vol 66 (5) ◽

Cited By ~ 11

Author(s):

Harmesh Sahay ◽

Surendra Singh ◽

Rajeev Kaushik ◽

Anil Saxena ◽

Dilip Arora

Keyword(s):

16S Rdna ◽

Brackish Water ◽

Phylogenetic Analyses ◽

Halophilic Bacteria ◽

Phenotypic Characterization ◽

Rrna Gene ◽

Polymorphism Analysis ◽

Gram Positive Bacteria ◽

Nutrient Agar Medium

AbstractCulture dependent phenotypic characterization and 16S rDNA based phylogenetic analyses were applied to study the aerobic halophilic bacterial population present in the Pulicat brackish-water Lake of India. Five different media were employed for isolation of bacteria. A total of 198 morphotypes were recovered, purified and screened for salt tolerance in nutrient agar medium amended with 5–25% NaCl. Based on 16S rDNA restriction fragment length polymorphism analysis with three restriction endonucleases, 51 isolates tolerant to 5% or more NaCl were grouped into 29 clusters. Phylogenetic analysis using 16S rRNA gene sequences revealed that 29 strains could further be allocated into two clades: 19 to Firmicutes and 10 to γ-Proteobacteria. Firmicutes included low G+C Gram-positive bacteria related to family Bacillaceae, which included five genera Bacillus, Virgibacillus, Rummelibacillus, Alkalibacillus and Halobacillus. Another genera included in Firmicutes was Salimicrobium halophilum. In the γ-Proteobacteria group, all the isolates belonged to one genus Halomonas, represented by six different species Halomonas salina, H. shengliensis, H. salifodinae, H. pacifica, H. aquamarina and H. halophila. Most of the isolates exhibited cellulase, xylanase, amylase and protease activities.

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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 Two Distinct Phytoplasma Species, ‘Candidatus Phytoplasma cynodontis’ and ‘Candidatus Phytoplasma asteris,’ Simultaneously Associated with Yellow Decline of Wodyetia bifurcata (Foxtail Palm) in Malaysia

Plant Disease ◽

10.1094/pdis-04-13-0412-pdn ◽

2013 ◽

Vol 97 (11) ◽

pp. 1504-1504 ◽

Cited By ~ 7

Author(s):

N. Naderali ◽

N. Nejat ◽

Y. H. Tan ◽

G. Vadamalai

Keyword(s):

16S Rdna ◽

Nested Pcr ◽

Rflp Analysis ◽

Rrna Gene ◽

Gene Sequences ◽

First Report ◽

White Leaf ◽

General Decline ◽

Pcr Products ◽

Plant Pathol

The foxtail palm (Wodyetia bifurcata), an Australian native species, is an adaptable and fast-growing landscape tree. The foxtail palm is most commonly used in landscaping in Malaysia. Coconut yellow decline (CYD) is the major disease of coconut associated with 16SrXIV phytoplasma group in Malaysia (1). Symptoms consistent with CYD, such as severe chlorosis, stunting, general decline, and death were observed in foxtail palms from the state of Selangor in Malaysia, indicating putative phytoplasma infection. Symptomatic trees loses their green and vivid appearance as a decorative and landscape ornament. To determine the presence of phytoplasma, samples were collected from the fronds of 12 symptomatic and four asymptomatic palms in September 2012, and total DNA was extracted using the CTAB method (3). Phytoplasma DNA was detected in eight symptomatic palms using nested PCR with universal phytoplasma 16S rDNA primer pairs, P1/P7 followed by R16F2n/R16R2 (2). Amplicons (1.2 kb in length) were generated from symptomatic foxtail palms but not from symptomless plants. Phytoplasma 16S rDNAs were cloned using a TOPO TA cloning kit (Invitrogen). Several white colonies from rDNA PCR products amplified from one sample with R16F2n/R16R2 were sequenced. Phytoplasma 16S rDNA gene sequences from single symptomatic foxtail palms showed 99% homology with a phytoplasma that causes Bermuda grass white leaf (AF248961) and coconut yellow decline (EU636906), which are both members of the 16SrXIV ‘Candidatus Phytoplasma cynodontis’ group. The sequences also showed 99% sequence identity with the onion yellows phytoplasma, OY-M strain, (NR074811), from the ‘Candidatus Phytoplasma asteris’ 16SrI-B subgroup. Sequences were deposited in the NCBI GenBank database (Accession Nos. KC751560 and KC751561). Restriction fragment length polymorphism (RFLP) analysis was done on nested PCR products produced with the primer pair R16F2n/R16R2. Amplified products were digested separately with AluI, HhaI, RsaI, and EcoRI restriction enzymes based on manufacturer's specifications. RFLP analysis of 16S rRNA gene sequences from symptomatic plants revealed two distinct profiles belonging to groups 16SrXIV and 16SrI with majority of the 16SrXIV group. RFLP results independently corroborated the findings from DNA sequencing. Additional virtual patterns were obtained by iPhyclassifier software (4). Actual and virtual patterns yielded identical profiles, similar to the reference patterns for the 16SrXIV-A and 16SrI-B subgroups. Both the sequence and RFLP results indicated that symptoms in infected foxtail palms were associated with two distinct phytoplasma species in Malaysia. These phytoplasmas, which are members of two different taxonomic groups, were found in symptomatic palms. Our results revealed that popular evergreen foxtail palms are susceptible to and severely affected by phytoplasma. To our knowledge, this is the first report of a mixed infection of a single host, Wodyetia bifurcata, by two different phytoplasma species, Candidatus Phytoplasma cynodontis and Candidatus Phytoplasma asteris, in Malaysia. References: (1) N. Nejat et al. Plant Pathol. 58:1152, 2009. (2) N. Nejat et al. Plant Pathol. J. 9:101, 2010. (3) Y. P. Zhang et al. J. Virol. Meth. 71:45, 1998. (4) Y. Zhao et al. Int. J. Syst. Evol. Microbiol. 59:2582, 2009.

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First Report of Bituminaria Witches'-Broom in Australia Caused by a 16SrII Phytoplasma

Plant Disease ◽

10.1094/pdis-10-10-0738 ◽

2011 ◽

Vol 95 (2) ◽

pp. 226-226 ◽

Cited By ~ 9

Author(s):

N. Aryamanesh ◽

A. M. Al-Subhi ◽

R. Snowball ◽

G. Yan ◽

K. H. M. Siddique

Keyword(s):

Canary Islands ◽

Western Australia ◽

Nested Pcr ◽

Perfect Match ◽

Universal Primers ◽

Healthy Plant ◽

Rrna Gene ◽

Polymorphism Analysis ◽

First Report ◽

Two Samples

Bituminaria bituminosa (L.) Stirt. is a perennial legume known as Arabian pea that is used as a forage in arid areas and for stabilization of degraded soils. It is widely distributed in the Mediterranean Basin with wider adaptation across the Canary Islands (4). In July 2010, during a survey for phytoplasma, some Canary Island B. bituminosa plants with typical phytoplasma symptoms, including stunted growth with small leaves, shortened internodes, and bushy growth, were found in seed multiplication nurseries at Medina, Perth, Western Australia (115°48.5′E; 32°13.2′S). Two samples from plants with clear disease symptoms and two visibly healthy plants were collected and total DNA was extracted with the Illustra DNA extraction kit Phytopure (GE Healthcare) according to the manufacturer's instructions. Direct and nested PCR were used to test the presence of phytoplasma 16S rDNA in samples with universal primers P1/P7 and R16F2n/R16R2, respectively (1,3). The PCR amplifications from all diseased samples yielded an expected product of 1.8 kb by direct and 1.2 kb by nested PCR, but not from the healthy plant samples. The direct PCR product was used as a template DNA in sequencing and the DNA sequence was deposited in the NCBI GenBank (Accession No. HQ404357). Sequence homology analysis indicated there was a perfect match between the two isolates. BLAST search of the NCBI GenBank revealed that B. bituminosa phytoplasma shares >99% sequence identity with Crotalaria witches'-broom phytoplasma (Accession No. EU650181.1), pear decline phytoplasma (Accession No. EF656453.1), and Scaevola witches'-broom phytoplasma (Accession No. AB257291.1). On the basis of BLAST analyses of 16S rRNA gene sequences, B. bituminosa phytoplasma in Western Australia appears to belong to the peanut witches'-broom group (16SrII-D) of phytoplasma. Restriction fragment length polymorphism analysis was also performed on nested PCR products of two samples of B. bituminosa phytoplasma by separate digestion with HaeIII, Hind6I, HpaII, MboI, RsaI, Tru9I, and T-HB8I restriction enzymes. Samples yielded patterns similar to alfalfa witches'-broom phytoplasma (Accession No. AF438413) belonging to subgroup 16SrII-D (2). To our knowledge, this is the first report of a phytoplasma of the 16SrII-D group infecting B. bituminosa in Australia and should be referred to as “Bituminaria witches'-broom phytoplasma” (BiWB). This report also indicates that the occurrence of the phytoplasma in B. bituminosa may be widespread in the Canary Islands and other species of Bituminaria might be susceptible to infection by Bituminaria witches'-broom phytoplasma. References: (1) D. E. Gundersen and I.-M. Lee. Phytopathol. Mediterr. 35:144, 1996. (2) A. J. Khan et al. Phytopathology 92:1038, 2002. (3) I.-M. Lee et al. Int. J. Syst. Evol. Microbiol. 54:337, 2004. (4) P. Mendez et al. Grassland Sci. Eur. 11:300, 2006.

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First Report of Boll Rot of Cotton Caused by Pantoea agglomerans in China

Plant Disease ◽

10.1094/pdis-92-9-1364b ◽

2008 ◽

Vol 92 (9) ◽

pp. 1364-1364 ◽

Cited By ~ 10

Author(s):

Y. Z. Ren ◽

Y. Q. Liu ◽

S. L. Ding ◽

G. Y. Li ◽

H. Zhang

Keyword(s):

16S Rdna ◽

The United States ◽

Cell Suspensions ◽

Pantoea Agglomerans ◽

Universal Primers ◽

Rrna Gene ◽

Disease Symptoms ◽

First Report ◽

Boll Rot ◽

Cotton Bolls

Since the summer of 2006, bacterial boll rot of cotton has been observed on fruits of ‘Xinluzao 31’ (Xinluzao 6 × Acala) in Xinjiang Province. It resulted in as much as 20% yield loss in several fields. Symptoms do not appear on the outer carpel. In the infected cotton bolls, fibers do not mature completely and seed tissue exhibits brown necrotic coloration. Lint and seeds from 24 surface-disinfested cotton bolls were triturated and plated onto King's medium B (KB). Plates were incubated at 28°C for 48 h. Forty eight strains with yellow pigmentation on KB were characterized. All were nonfluorescent on KB, gram negative, facultatively anaerobic, unable to produce indole from tryptophan, able to reduce nitrate to nitrite, and produce acid from glucose, cellobiose, lactose, melibiose, and melonate. In addition, 16S rDNA in seven strains was amplified with universal primers (1). The PCR products were cloned into pGEM-T easy vector and sequenced. A BLAST search of the seven sequences against the GenBank nucleotide library indicated 100% identity with the 16S rDNA sequence of Enterobacter agglomerans strain A80. Then an additional primer pair, pagF and pagR (3), was used for more specific amplification of Pantoea agglomerans 16S rDNA, which resulted in single highly specific fragments of approximately 1 kb. On the basis of morphological, physiological, biochemical characteristics, and 16S rRNA gene sequence analysis, we identified the bacterium to be P. agglomerans. To confirm pathogenicity, cell suspensions (1 × 108 CFU/ml) of eight representative strains were used to inoculate cotton at peak bolling stage in the field. Cell suspensions, or water as the control, were applied to stigma scars, wall sutures, and scratch wounds on bracts, calyxes, and bolls. Alternatively, a needle was used to puncture through a drop of suspension placed on the boll wall suture and bracts. At least 20 bolls or flowers were inoculated with each bacterial strain per inoculation method. Infection occurred only when bacterial injections breached the endocarp of the boll either through the carpel wall or a suture between carpel sections. Disease symptoms developed 1 week postinoculation. The inoculated organism was reisolated from the diseased tissues. P. agglomerans is generally regarded to be a soil saprophyte or leaf epiphyte, but strains can opportunistically infect plants triggering gall formations or human wounds causing septic arthritis. The disease symptoms and pathogen characteristics observed in this study are identical to those reported in the United States (2). To our knowledge, this is the first report of P. agglomerans causing boll rot of cotton in China. References: (1) S. Manulisi and I. Barash. Mol. Plant Pathol. 4:307, 2003. (2) E. G. Medrano et al. J. Appl. Microbiol. 103:436, 2007. (3) S. Vorwerk et al. Agric. For. Entomol. 9:57, 2007.

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