scholarly journals First Report of Syringa oblata and S. reticulata Leafroll Disease in China

Plant Disease ◽  
2009 ◽  
Vol 93 (3) ◽  
pp. 322-322
Author(s):  
Z. N. Li ◽  
H. Min ◽  
Y. Yan ◽  
Z. Zhao ◽  
W. J. Wu ◽  
...  
Keyword(s):  
16S Rdna ◽  
Rflp Analysis ◽  
Universal Primers ◽  
University Campus ◽  
Sequence Comparisons ◽  
16S Rdna Sequence ◽  
First Report ◽  
Pcr Products ◽  
Leafroll Disease ◽  
Total Dna

Syringa oblata is an important ornamental tree widely grown in China. In September of 2008, S. oblata plants exhibiting symptoms of leafroll and yellowing were found in a garden on the Northwest A&F University campus. Samples were collected from this site. Total DNA was extracted from 0.5 g of phloem tissue from leaf midribs and stems of each sample. DNA samples were analyzed with a nested PCR assay using phytoplasma 16S rDNA universal primers R16mF2/R16mR1 followed by specific primers R16F2n/R16R2 (1), which amplified a 1,452- and 1,246-bp product, respectively. We tested all 30 lilac samples, 20 of which had symptoms of leafroll and yellowing. These produced the expected 1,452- and 1,246-bp PCR products In contrast, the remaining 10 samples from symptomless trees yielded no PCR products. We also surveyed another lilac variety (Syringa reticulata), which is widely grown on the campus, and tested 50 samples with the above method. Again, 1.4- and 1.2-kb PCR products were amplified from all 30 trees displaying leafroll and yellowing symptoms, but not from the other 20 samples from symptomless trees. A comparative analysis of sequences derived from the two hosts showed that the phytoplasmas infecting them were most similar (>99%) to paulownia witches'-broom (PaWB) phytoplasma (GenBank Accession No. EF199937). Restriction fragment length polymorphism (RFLP) analysis of the nested 1.2-kb 16S rDNA products with endonucleases AluI and MseI indicated that all symptomatic plants were infected by the phytoplasmas belonging to aster yellow group (16SrI) subgroup D (16SrI-D) PaWB phytoplasma (2). 16S rDNA sequence comparisons and RFLP analysis of the cloned 16S rDNA from S. oblata (GenBank Accession No. FJ445224) and S. reticulate (GenBank Accession No. FJ445225) indicated that the phytoplasmas infecting them were nearly identical (99.8% identity). To our knowledge, this is the first report of the presence of the phytoplasma associated with a leafroll disease of S. oblata and S. reticulata in China. References: (1) D. E. Gundersen and I.-M. Lee. Phytopathol. Mediterr. 35:144, 1996. (2) I.-M. Lee et al. Int. J. Syst. Bacteriol. 48:1153, 1998.

scholarly journals First Report of ‘Candidatus Phytoplasma asteris’-Related Strains Infecting Lily in Mexico

Plant Disease ◽  
2008 ◽  
Vol 92 (6) ◽  
pp. 979-979 ◽  
Author(s):  
N. E. Cortés-Martínez ◽  
E. Valadez-Moctezuma ◽  
L. X. Zelaya-Molina ◽  
N. Marbán-Mendoza
Keyword(s):  
16S Rdna ◽  
The Czech Republic ◽  
First Report ◽  
Rdna Sequences ◽  
Flower Abortion ◽  
16S Rdna Sequences ◽  
Pcr Products ◽  
Total Dna ◽  
Nested Pcr Assays ◽  
Symptomatic Plant

In recent years, lily (Lilium spp.) has become an important ornamental crop in diverse regions of Mexico. Since 2005, unusual symptoms have been observed on lily plants grown from imported bulbs in both greenhouse and production plots at San Pablo Ixayo, Boyeros, and Tequexquinauac, Mexico State. Symptoms included a zigzag line pattern on leaves, dwarfism, enlargement of stems, shortened internodes, leaves without petioles growing directly from bulbs, air bulbils, death of young roots, atrophy of flower buttons, and flower abortion. Symptoms were experimentally reproduced on healthy lily plants by graft inoculation. Total DNA was extracted from 50 diseased, 10 symptomless, and 10 graft-inoculated plants by the method of Dellaporta et al. (2). DNA samples were analyzed for phytoplasma presence by two different nested PCR assays. One assay employed ribosomal RNA gene primer pair P1/P7 followed by R16F2n/R16R2 (1), whereas ribosomal protein (rp) gene primer pairs rpF1/rpR1 and rp(I)F1A/rp(I)R1A (4) were used in a second assay. A DNA fragment approximately 1.2 kb long was consistently amplified from all symptomatic plant samples only by both assays. A comparative analysis of 16S rDNA sequences (Genbank Accession Nos. EF421158–EF421160 and EU124518–EU124520) and rp gene sequences (EU277012–EU277014), derived from PCR products, revealed that phytoplasma infecting lily were most similar (99.9% to 16S rDNA and 99.7% to rp) to carrot phytoplasma sp. ca2006/5 and also were similar (99.8% to 16SrDNA and 99.2% to rp) to broccoli phytoplasma sp. br273. Both carrot and broccoli phytoplasmas were classified as members of aster yellow 16S rDNA restriction fragment length polymorphism subgroup 16SrI-B (3). Although infection of lilies by aster yellows (‘Ca. phytoplasma asteris’) subgroup 16SrI-B and 16SrI-C was reported from the Czech Republic and Poland, to our knowledge, this is the first report of ‘Ca. phytoplasma asteris’-related strains associated with lily plants in Mexico. References: (1) R. F. Davis et al. Microbiol. Res. 158:229, 2003. (2) S. L. Dellaporta et al. Plant Mol. Biol. Rep. 1:19, 1983. (3) B. Duduk et al. Bull. Insectol. 60 2:341, 2007. (4) I.-M. Lee et al. Int. J. Syst. Evol. Microbiol. 54:337, 2004.

scholarly journals First Report of Group 16SrXII Phytoplasma Causing Stolbur Disease in Potato Plants in the Eastern and Southern Anatolia Regions of Turkey

Plant Disease ◽  
2010 ◽  
Vol 94 (11) ◽  
pp. 1374-1374 ◽  
Author(s):  
S. Eroglu ◽  
H. Ozbek ◽  
F. Sahin
Keyword(s):  
16S Rdna ◽  
Rdna Sequence ◽  
Universal Primers ◽  
Effective Control ◽  
Diseased Plant ◽  
Eastern Anatolia ◽  
Universal Primer ◽  
16S Rdna Sequence ◽  
First Report ◽  
Potato Plants

In recent years, a stolbur-like disease has had devastating effects on the yield and marketable quality of potato production in Erzurum (Eastern Anatolia) and Akcakale-Sanliurfa (Southern Anatolia) regions of Turkey. Potato plants exhibited several different symptoms including stunting, upward rolling of the top leaves along with reddish or purplish coloration, chlorosis, shortened internodes, swollen nodes, proliferated axillary buds, aerial tubers, and early plant decline. An extensive survey from 2003 to 2010 was performed and diseased plant samples were collected. Total genomic DNAs were isolated from the leaf mid-veins of the six different symptomatic and two symptomless plants selected. Nested-PCRs, carried out by using phytoplasma-universal primer pair P1/P7 followed by R16F2n/R16R2 (2), amplified 16S rDNA fragments (F2nR2) from only templates derived from symptomatic plants. F2nR2 PCR products from two independent symptomatic plants were cloned and sequenced from both directions with M13 universal primers. The obtained 16S rDNA sequence (GenBank Accession No HM485579) was subjected to virtual restriction fragment length polymorphism (RFLP) analysis using iphyclassifier software (3). Results indicated that the phytoplasma, here identified in association with potato plants, shared best sequence identity (99%) with members of subgroup 16SrXII-A (e.g., GenBank Accession No. EU010006). Moreover, collective RFLP pattern of potato-associated phytoplasma differed from digestion profiles of previously described 16SrXII subgroups, sharing best similarity coefficient (0.94) with the reference phytoplasma strain of subgroup 16SrXII-A (GenBank Accession No. AJ964960). Thus, it was confirmed that potato-associated phytoplasma represents a new 16SrXII subgroup (16SrXII-N). Furthermore, a new primer set (PatsecF/PatsecR) was designed for priming specific PCR-amplification of potato-associated phytoplasma 16S rDNA sequence. PCR reaction was successfully used for specifically detecting stolbur phytoplasma in infected potato plants. The use of this method may help to determine possible alternative hosts and vectors of potato phytoplasma, which is important for development of an integrated management strategy for effective control of this disease in the future. Presence of potato stolbur diseases in the Eastern Anatolia Region of Turkey has previously been reported (1). To our knowledge, this is the first report of occurrence of a 16SrXII group phytoplasma causing potato stolbur diseases caused in the Eastern and Southern Anatolia regions of Turkey. References: (1) A. Citir. J. Turk. Phytopathol. 14:53, 1985. (2) D. E. Gundersen and I. M. Lee. Phytopathol. Mediterr. 35:144, 1996. (3)Y. Zhao et al. Int. J. Syst. Evol. Microbiol. 59:2582, 2009.

scholarly journals 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 ◽  
2013 ◽  
Vol 97 (11) ◽  
pp. 1504-1504 ◽  
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.

scholarly journals A Phytoplasma Closely Related to the Pigeon Pea Witches'-Broom Phytoplasma (16Sr IX) Is Associated with Citrus Huanglongbing Symptoms in the State of São Paulo, Brazil

2008 ◽  
Vol 98 (9) ◽  
pp. 977-984 ◽  
Author(s):  
D. C. Teixeira ◽  
N. A. Wulff ◽  
E. C. Martins ◽  
E. W. Kitajima ◽  
R. Bassanezi ◽  
...  
Keyword(s):  
16S Rdna ◽  
External Source ◽  
Pigeon Pea ◽  
Sao Paulo ◽  
Rdna Sequence ◽  
Universal Primers ◽  
Insect Vector ◽  
São Paulo ◽  
16S Rdna Sequence ◽  
Pcr Products

In February 2007, sweet orange trees with characteristic symptoms of huanglongbing (HLB) were encountered in a region of São Paulo state (SPs) hitherto free of HLB. These trees tested negative for the three liberibacter species associated with HLB. A polymerase chain reaction (PCR) product from symptomatic fruit columella DNA amplifications with universal primers fD1/rP1 was cloned and sequenced. The corresponding agent was found to have highest 16S rDNA sequence identity (99%) with the pigeon pea witches'-broom phytoplasma of group 16Sr IX. Sequences of PCR products obtained with phytoplasma 16S rDNA primer pairs fU5/rU3, fU5/P7 confirm these results. With two primers D7f2/D7r2 designed based on the 16S rDNA sequence of the cloned DNA fragment, positive amplifications were obtained from more than one hundred samples including symptomatic fruits and blotchy mottle leaves. Samples positive for phytoplasmas were negative for liberibacters, except for four samples, which were positive for both the phytoplasma and ‘Candidatus Liberibacter asiaticus’. The phytoplasma was detected by electron microscopy in the sieve tubes of midribs from symptomatic leaves. These results show that a phytoplasma of group IX is associated with citrus HLB symptoms in northern, central, and southern SPs. This phytoplasma has very probably been transmitted to citrus from an external source of inoculum, but the putative insect vector is not yet known.

scholarly journals First Report of Herbaspirillum rubrisubalbicans Causing Mottled Stripe Disease on Sugarcane in China

Plant Disease ◽  
2010 ◽  
Vol 94 (3) ◽  
pp. 379-379 ◽  
Author(s):  
ZQ. Tan ◽  
R. Men ◽  
RY. Zhang ◽  
Z. Huang
Keyword(s):  
16S Rdna ◽  
Uv Light ◽  
Rdna Sequence ◽  
Universal Primers ◽  
Bacterial Cells ◽  
Bacterial Strains ◽  
Potato Dextrose Agar Medium ◽  
16S Rdna Sequence ◽  
First Report ◽  
Herbaspirillum Rubrisubalbicans

Narrow, red stripes were observed on leaves and sheaths of sugarcane in 2007 in DanZhou County of Hainan Province and XuWen County of GuangDong Province, China. Stripes were parallel to the leaf veins. Some stripes were short (2 to 10 cm) and some were >1 m long, extending from the base of leaves. Width of the stripes was 2 to 4 mm. Symptoms varied with the cultivar. Cv. Taiwang 25, which was the most affected, exhibited red stripes and stalk death from the apex. Cvs. Taiwang 26 and Guang Dong 00236 were slightly affected with only red stripes. Symptoms on cv. Taiwang 22 were mottled stripes. Severe losses were observed in the infected fields that were planted with cv. Taiwang 25, but there were no obvious losses in fields planted with the other three cultivars. Isolations were made from 10 individual plants from different cultivars and provinces that had red stripes, two of which also had apex death. Five independent bacterial isolates were obtained from tissue showing the red stripe symptoms on potato dextrose agar medium. The percentage of positive samples was 50%. No bacteria were obtained from necrotic apex tissue. Bacterial cells were 0.92 to 1.55 × 0.20 to 0.22 μm slightly curved rods that were motile with one to two polar flagella. Colonies on nutrient agar were 2 to 3 mm in diameter, circular, smooth, entire, and milky white. Colonies on King's medium B were nonfluorescent under 365-nm UV light. Five bacterial strains were inoculated by injecting bacterial suspensions (1 × 108 CFU/ml) into the base of the leaves of 6-month-old cv. Taiwang 25 plants (1). Red stripes appeared 7 to 10 days after inoculation and bacteria were reisolated. The reisolated bacteria were identical to the original strains in colony morphology and 16S rDNA sequence. A hypersensitive response appeared within 24 h when 1 × 108 CFU/ml bacteria suspensions were infiltrated into tobacco leaves. Approximately 1,000-bp DNA fragments were amplified with universal primers UP1 (5′-TACGTGCCAGCAGCCGCGGTAATA-3′) and UP2 (5′-AGTAAGGAGGGTATCCAACCGCA-3′) (3). Primers UP1 and UP2 are complementary to nucleotide sequence 509 to 523 and 1541 to 1522, respectively, of the Escherichia coli 16S rDNA gene. The fragment amplified by these primers was approximately 1,032 bp. The 16S rDNA sequences of the five strains were deposited in GenBank as Accession Nos. GQ476791–5. They all shared 99% nucleotide identity with the type strain of Herbaspirillum rubrisubalbicans (GenBank No. AJ238356.1). All five strains were identified as H. rubrisubalbicans on the basis of 16S rDNA sequence and pathogenicity to sugarcane, and the disease was identified as mottled stripe disease (2). Since we were not able to isolate bacteria from necrotic apex tissue, this symptom on cv. Taiwang 25 may not be related to the H. rubrisubalbicans infection. To our knowledge, this is the first report of mottled stripe disease in China. References: (1) H. M. A. EI-Komy et al. Folia Microbiol. 48:787, 2003. (2) A. S. Saumtally et al. A Guide to Sugarcane Diseases. P. Rott et al., eds. CIRAD and ISSCT, Montpellier, France, 2000. (3) Yan Zhi Yong et al. Chin. J. Epidemiol. 24:296, 2003.

scholarly journals First Report of a Geminivirus Inducing Yellow Mottle in Okra (Abelmoschus esculentus) in Mexico

Plant Disease ◽  
2003 ◽  
Vol 87 (2) ◽  
pp. 202-202
Author(s):  
R. De La Torre-Almaráz ◽  
A. C. Monsalvo-Reyes ◽  
R. F. Rivera-Bustamante ◽  
J. Méndez-Lozano
Keyword(s):  
Blot Hybridization ◽  
Fruit Production ◽  
Dot Blot ◽  
Sequence Comparisons ◽  
First Report ◽  
Squash Leaf Curl Virus ◽  
Pcr Products ◽  
Yellow Mottle ◽  
Total Dna ◽  
Leaf Curl Virus

Okra is an annual vegetable species native to Africa. In Mexico, the states of Tamaulipas, Guerrero, and Morelos contain the most important okra-producing areas. Viral-like diseases have recently affected the fruit production. In the field, the most common symptoms encountered include yellow streak, distortion of fruits, and foliar yellow mottle. Total DNA extracts from symptomatic okra plants were used as a template for polymerase chain reaction (PCR)-based detection using begomovirus-specific primers. RepMot and CPMot primers (1) were used for the amplification of DNA fragments that included the Rep and coat protein (CP) genes of begomoviruses. PCR results suggested the presence of a begomovirus in symptomatic plants. Southern and dot blot hybridization analysis were performed using a DNA fragment containing the CP gene of Pepper huasteco virus as a probe. Hybridization conducted under low stringency conditions confirmed the presence of a geminivirus. Additionally, transmission by grafting and biolistic (total DNA extracts from symptomatic plants) inoculations induced consistently severe streak fruits and yellow mottle symptoms in okra plants. Cloning of the PCR products (approximately 632-bp fragment) was performed in the PCRTopo vector (Invitrogen, San Diego, CA). Cloned viral inserts were sequenced (Genbank Accession No. AF349113). Nucleotide sequence comparisons were performed using the Clustal Method (MegAlign, DNAStar software, Madison, WI) with the GenBank database. Analysis of the PCR products confirmed the begomovirus nature of the sequence. The first 64 amino acids of the CP had 89% identity with Squash leaf curl virus while the intergenic region had 85% identity with Sida golden mosaic virus. To our knowledge, this is the first report of a begomovirus infecting okra in Mexico. Reference: 1) J. T. Ascencio et al. Plant Dis. 86:692, 2002.

scholarly journals First Report of Paulownia Witches'-Broom Phytoplasma in China

Plant Disease ◽  
2008 ◽  
Vol 92 (7) ◽  
pp. 1134-1134 ◽  
Author(s):  
H. N. Yue ◽  
Y. F. Wu ◽  
Y. Z. Shi ◽  
K. K. Wu ◽  
Y. R. Li
Keyword(s):  
16S Rdna ◽  
Sequence Data ◽  
Rflp Analysis ◽  
Shaanxi Province ◽  
Economic Losses ◽  
Rrna Gene ◽  
First Report ◽  
Ctab Method ◽  
Phytoplasma Infection ◽  
Total Dna

Paulownia witches'-broom (PaWB) is one of the most important diseases affecting Paulownia tomentosa trees in China. According to 2006 statistics, the disease has affected 880,000 ha of trees for timber production causing billions of dollars in economic losses. During the spring and summer of 2006, a survey was done in Shaanxi Province to confirm phytoplasma infection of paulownia trees exhibiting symptoms of witches'-broom, stunting, yellowing, and proliferating secondary shoots. Foliage samples were collected from 24 symptomatic and 8 symptomless paulownia plants in eight different production fields. 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 1.4-kb and a 1.2-kb product, respectively, from symptomatic plants. Restriction fragment length polymorphism (RFLP) analysis of the nested 1.2-kb 16S rDNA products with AluI, MseI, HhaI, HpaI, RsaI, BfaI, HinfI, and TaqI endonuclease (2) indicated that all symptomatic plants were infected by a phytoplasma belonging to aster yellows group (16SrI) subgroup D (16SrI-D) phytoplasma strains. A 1.2-kb 16S rDNA sequence (GenBank Accession No. DQ851169) derived from representative strain PaWB-Shaanxi was identical (100%) to that of PaWB phytoplasma (L27033), a known subgroup 16SrI-D strain from Taiwan (2). The agreement between the RFLP analysis and sequence data confirms that PaWB from Shaanxi is a member of subgroup 16SrI-D. To our knowledge, this is the first report of PaWB disease being present in China and of its association with the 16SrI-D subgroup. 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.

scholarly journals First Report of a Group 16SrII Phytoplasma Infecting Chickpea in Oman

Plant Disease ◽  
2006 ◽  
Vol 90 (7) ◽  
pp. 973-973 ◽  
Author(s):  
N. A. Al-Saady ◽  
A. M. Al-Subhi ◽  
A. Al-Nabhani ◽  
A. J. Khan
Keyword(s):  
Nested Pcr ◽  
Animal Feed ◽  
Restriction Enzymes ◽  
Universal Primers ◽  
Polymorphism Analysis ◽  
Disease Symptoms ◽  
First Report ◽  
Pcr Products ◽  
Polymerase Chain ◽  
Group 2

Chickpea (Cicer arietinum), locally known as “Dungo”, is grown for legume and animal feed mainly in the interior region of Oman. During February 2006, survey samples of chickpea leaves from plants showing yellows disease symptoms that included phyllody and little leaf were collected from the Nizwa Region (175 km south of Muscat). Total nucleic acid was extracted from asymptomatic and symptomatic chickpea leaves using a cetyltrimethylammoniumbromide method with modifications (3). All leaf samples from eight symptomatic plants consistently tested positive using a polymerase chain reaction assay (PCR) with phytoplasma universal primers (P1/P7) that amplify a 1.8-kb phytoplasma rDNA product and followed by nested PCR with R16F2n/R16R2 primers yielding a product of 1.2 kb (2). No PCR products were evident when DNA extracted from healthy plants was used as template. Restriction fragment length polymorphism analysis of nested PCR products by separate digestion with Tru9I, HaeIII, HpaII, AluI, TaqI, HhaI, and RsaI restriction enzymes revealed that a phytoplasma belonging to group 16SrII peanut witches'-broom group (2) was associated with chickpea phyllody and little leaf disease in Oman. Restriction profiles of chickpea phytoplasma were identical with those of alfalfa witches'-broom phytoplasma, a known subgroup 16SrII-B strain (3). To our knowledge, this is the first report of phytoplasma infecting chickpea crops in Oman. References: (1) A. J. Khan et al. Phytopathology, 92:1038, 2002. (2). I.-M. Lee et al. Int. J. Syst. Bacteriol. 48:1153, 1998 (3) M. A. Saghai-Maroof et al. Proc. Natl. Acad. Sci. USA. 81:8014, 1984.

scholarly journals First Report of an Aster Yellows Phytoplasma Associated with Cabbage in Southern Texas

Plant Disease ◽  
2001 ◽  
Vol 85 (4) ◽  
pp. 447-447 ◽  
Author(s):  
I.-M. Lee ◽  
R. A. Dane ◽  
M. C. Black ◽  
Noel Troxclair
Keyword(s):  
16S Rdna ◽  
Nested Pcr ◽  
Restriction Enzymes ◽  
Diagnostic Procedures ◽  
Acid Extraction ◽  
Insect Vector ◽  
Aster Yellows ◽  
First Report ◽  
Pcr Products ◽  
Aster Yellows Phytoplasma

In early spring 2000 carrot crops in southwestern Texas were severely infected by an outbreak of phyllody associated with aster yellows phytoplasma. Cabbage crops that had been planted adjacent to these carrot fields began to display previously unobserved symptoms characteristic of phytoplasma infection. Symptoms included purple discoloration in leaf veins and at the outer edges of leaves on cabbage heads. Proliferation of sprouts also occurred at the base of the stem and between leaf layers of some plants, and sprouts sometimes continued to proliferate on extended stems. About 5% of cabbage plants in the field exhibited these symptoms. Two symptomless and four symptomatic cabbage heads were collected in early April from one cabbage field. Veinal tissues were stripped from each sample and used for total nucleic acid extraction. To obtain specific and sufficient amount of PCR products for analysis, nested PCR was performed by using primer pairs (first with P1/P7 followed by R16F2n/R16R2) (1,2) universal for phytoplasma detection. A specific 16S rDNA fragment (about 1.2 kb) was strongly amplified from the four symptomatic but not from the two asymptomatic samples. The nested PCR products obtained from the four symptomatic samples were then analyzed by restriction fragment length polymorphism (RFLP) using the restriction enzymes MseI, HhaI, and HpaII, and the RFLP patterns were compared to the published patterns of known phytoplasmas (1). The resulting RFLP patterns were identical to those of a phytoplasma belonging to subgroup B of the aster yellows phytoplasma group (16SrI). These RFLP patterns were also evident in putative restriction sites observed in a 1.5 kbp nucleotide sequence of the 16S rDNA. This is the first report of aster yellows phytoplasma associated disease symptoms in cabbage in Texas. The occurrence of cabbage proliferation coincided with the presence of high populations of the insect vector, aster leafhopper. References: (1) I.-M. Lee et al. Int. J. Syst. Bacteriol. 48:1153, 1998. (2) B. Schneider et al. 1995. Molecular and Diagnostic Procedures in Mycoplasmology, Vol. I. Academic Press, San Diego, CA.

scholarly journals Fast and reliable PCR/sequencing/RFLP assay for identification of fungi in onychomycoses

2006 ◽  
Vol 55 (9) ◽  
pp. 1211-1216 ◽  
Author(s):  
Michel Monod ◽  
Olympia Bontems ◽  
Christophe Zaugg ◽  
Barbara Léchenne ◽  
Marina Fratti ◽  
...  
Keyword(s):  
Rflp Analysis ◽  
Fungal Species ◽  
Universal Primers ◽  
Cure Rate ◽  
Routine Identification ◽  
Identification Of Fungi ◽  
Pcr Products ◽  
Nail Sample ◽  
Fungal Dna ◽  
Rflp Assay

Fusarium spp. and other non-dermatophyte fungi are repeatedly isolated from abnormal nails. To investigate whether these fungi are the aetiological agents of infection or simply transient contaminants, a PCR/sequencing/RFLP assay was developed for direct and routine identification of the infecting fungi in onychomycosis. Fungal DNA was readily extracted using a commercial kit after dissolving nail fragments in a Na2S solution. Amplification of part of the 28S rDNA by PCR was performed with universal primers and the fungal species were identified by sequencing. The PCR/sequencing results were comparable with microbiological identification from the same nail sample. In addition to dermatophytes, Fusarium spp. and other less frequently isolated non-dermatophyte fungi were identified as single fungal agents in onychomycosis. Moreover, mixed infections were clearly demonstrated in 10 % of cases by RFLP analysis of PCR products. Identification of infectious agents could be obtained in 2 days, whilst results from fungal cultures take 1–3 weeks. Rapid and reliable molecular identification of the infectious fungus expedites the choice of appropriate antifungal therapy, thereby improving the cure rate of onychomycosis.

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