Witches' Broom Disease of Lime Contributes to Phytoplasma Epidemics and Attracts Insect Vectors.

An insect-transmitted phytoplasma causing Witches' Broom Disease of Lime (WBDL) is responsible for the drastic decline in lime production in several countries. However, it is unclear how WBDL phytoplasma (WBDLp) induces witches' broom symptoms and if these symptoms contribute to the spread of phytoplasma. Here we show that the gene encoding SAP11 of WBDLp (SAP11WBDL) is present in all WBDLp isolates collected from diseased trees. SAP11WBDL interacts with acid lime (Citrus aurantifolia) TCP transcription factors, specifically members of the TB1/CYC class that have a role in suppressing axillary branching in plants. Sampling of WBDLp-infected lime trees revealed that WBDLp titers and SAP11WBDL expression levels were higher in symptomatic leaves compared with asymptomatic sections of the same trees. Moreover, the witches' brooms were found to attract the vector leafhopper. Defense genes that have a role in plant defense responses to bacteria and insects are more downregulated in witches' brooms compared with asymptomatic sections of trees. These findings suggest that witches' broom-affected parts of the trees contribute to WBDL epidemics by supporting higher phytoplasma titers and attracting insect vectors.

Identification of genome regions controlling cotyledon, pod wall/seed coat and pod wall resistance to pea weevil through QTL mapping.

Pea weevil, Bruchus pisorum, is one of the limiting factors for field pea (Pisum sativum) cultivation in the world with pesticide application the only available method for its control. Resistance to pea weevil has been found in an accession of Pisum fulvum but transfer of this resistance to cultivated pea (P. sativum) is limited due to a lack of easy-to-use techniques for screening interspecific breeding populations. To address this problem, an interspecific population was created from a cross between cultivated field pea and P. fulvum (resistance source). Quantitative trait locus (QTL) mapping was performed to discover the regions associated with resistance to cotyledon, pod wall/seed coat and pod wall resistance. Three major QTLs, located on linkage groups LG2, LG4 and LG5 were found for cotyledon resistance explaining approximately 80 % of the phenotypic variation. Two major QTLs were found for pod wall/seed coat resistance on LG2 and LG5 explaining approximately 70 % of the phenotypic variation. Co-linearity of QTLs for cotyledon and pod wall/seed coat resistance suggested that the mechanism of resistance for these two traits might act through the same pathways. Only one QTL was found for pod wall resistance on LG7 explaining approximately 9 % of the phenotypic variation. This is the first report on the development of QTL markers to probe Pisum germplasm for pea weevil resistance genes. These flanking markers will be useful in accelerating the process of screening when breeding for pea weevil resistance.

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

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.

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

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.

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

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.

First Report of 16S rDNA II Group Phytoplasma on Polygala mascatense, a Weed in Oman.

Polygala mascatense Boiss. (family Polygalaceae) is a common weed found in neglected farms, under date palm trees, and in stony locations throughout the Sultanate of Oman (1). It is a perennial herb approximately 30 to 40 cm tall, has slender branches, is woody at the base, and has linear leaves with purple flowers. Recently (November 2004), in the interior region of Oman (210 km south of Muscat), some polygala plants were found stunted with small leaves, bushy growth, and the floral parts were showing phyllody symptoms. Total genomic DNA extracted from asymptomatic and symptomatic plants with modified cetyltrimethylammoniumbromide (CTAB) buffer method (4) was used as a template for direct polymerase chain reaction (PCR) amplification of phytoplasma 16S rDNA with P1/P7 primers. Direct PCR product was used as template DNA for nested PCR with primers R16F2n/R16R2. DNA from plants infected with alfalfa and lime witches'-broom phytoplasma was used as positive controls, and DNA from healthy plants and water was used as negative controls. Products from nested PCR (1.2 kb) were analyzed by using single endonuclease enzyme digestion (restriction fragment length polymorphism [RFLP]) with Tru9I, HaeIII, HhaI, TaqI, AluI, and RsaI (3). The results showed the presence of a 1.8-kb product amplified with direct PCR and a 1.2-kb product of the nested PCR from infected polygala and the positive controls, whereas no PCR products were observed in the negative controls. The PCR assay confirmed the presence of phytoplasma causing witches'-broom disease in polygala. The RFLP results showed the polygala phyto-plasma to be most similar to the alfalfa phytoplasma, a member of 16SrII group (2). Infected polygala weeds may serve as a reservoir for alfalfa witches'-broom phytoplasma that causes annual losses over $25 million to alfalfa cultivation in Oman (2). A detailed investigation needs to be carried out to establish transmission of phytoplasma from polygala to alfalfa. To our knowledge, this is the first report of phytoplasma infecting polygala weeds in Oman. References: (1) S. A. Ghazanfar. Pages 95-96 in: An Annotated Catalogue of the Vascular Plants in Oman. Scripta Botanica Belgica Meise, National Botanic Garden of Belgium, 1992. (2) A. J. Khan et al. Phytopathology 92:1038, 2002. (3) I. M. Lee et al. Int. J. Syst. Bacteriol. 1153, 1998. (4) M. A. Saghai-Maroof et al. Proc. Natl. Acad. Sci. USA 81:8014, 1984.

First Report of Witches'-Broom Disease of Sesame (Sesamum indicum) in Oman.

Sesame is the major oil seed crop in Oman. During 2004, disease symptoms were observed at Nizwa, 175 km south of Muscat. Symptoms included phyllody and excessive development of short shoots and internodes resulting in little leaves. Total genomic DNA was extracted from healthy and symptomatic plants with a modified cetyltrimethylammoniumbromide (CTAB) buffer method (2). DNA samples were assayed by polymerase chain reaction (PCR), with the 16S rDNA amplified using primers P1 and P7. Direct PCR products were used as template DNA for nested PCR with primers R16F2n and R16R2. Direct PCR products were analyzed by restriction fragment length polymorphism (RFLP) with four restriction enzymes, Tru9I, HaeIII, HhaI, and RsaI. DNAs from alfalfa and lime plants infected by witches'-broom phytoplasmas were used as positive controls and DNA from healthy plants and water were negative controls. The results showed the presence of a 1.8-kb product amplified with the direct PCR and a 1.2-kb product of the nested PCR from infected sesame and the positive controls. No PCR product was observed in the negative control. The PCR assay confirmed the presence of phytoplasma causing witches'-broom disease in sesame. The RFLP results showed the sesame phytoplasma to be most similar to the alfalfa phytoplasma, a member of 16SrII group (1). To our knowledge, this is the first report of a phytoplasma of the 16Sr II group causing witches'-broom disease on sesame in the Sultanate of 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.

Molecular identification of a new phytoplasma associated with alfalfa witches'-broom in oman.

ABSTRACT Alfalfa (Medicago sativa) plants showing witches'-broom symptoms typical of phytoplasmas were observed from Al-Batinah, Al-Sharqiya, Al-Bureimi, and interior regions of the Sultanate of Oman. Phytoplasmas were detected from all symptomatic samples by the specific amplification of their 16S-23S rRNA gene. Polymerase chain reaction (PCR), utilizing phytoplasma-specific universal primer pairs, consistently amplified a product of expected lengths when DNA extract from symptomatic samples was used as template. Asymptomatic plant samples and the negative control yielded no amplification. Restriction fragment length polymorphism profiles of PCR-amplified 16S-23S rDNA of alfalfa using the P1/P7 primer pair identified phytoplasmas belonging to peanut witches'-broom group (16SrII or faba bean phyllody). Restriction enzyme profiles showed that the phytoplasmas detected in all 300 samples belonged to the same ribosomal group. Extensive comparative analyses on P1/P7 amplimers of 20 phytoplasmas with Tru9I, Tsp509I, HpaII, TaqI, and RsaI clearly indicated that this phytoplasma is different from all the other phytoplasmas employed belonging to subgroup 16SrII, except tomato big bud phytoplasma from Australia, and could be therefore classified in subgroup 16SrII-D. The alfalfa witches'-broom (AlfWB) phytoplasma P1/P7 PCR product was sequenced directly after cloning and yielded a 1,690-bp product. The homology search showed 99% similarity (1,667 of 1,690 base identity) with papaya yellow crinkle (PapayaYC) phytoplasma from New Zealand. A phylogenetic tree based on 16S plus spacer regions sequences of 35 phytoplasmas, mainly from the Southern Hemisphere, showed that AlfWB is a new phytoplasma species, with closest relationships to PapayaYC phytoplasmas from New Zealand and Chinese pigeon pea witches'-broom phytoplasmas from Taiwan but distinguishable from them considering the different associated plant hosts and the extreme geographical isolation.

First Report of Alfalfa Witches Broom Disease in Oman Caused by a Phytoplasma of the 16Sr II Group.

Alfalfa (Medicago sativa L.) is a primary forage crop in the Sultanate of Oman. A new disease of alfalfa in Oman is characterized by proliferation of shoots and yellowing of leaves in 1- to 2-year-old plants and tillering of stems in 4- to 5-year-old plants. Annual losses due to this disease are estimated at more than US$ 23 million. Samples of healthy and infected alfalfa plants were collected from different regions. Total DNA was extracted according to Khadhair et al. (1), with minor modifications. Amplification of 16S rDNA was done using a nested polymerase chain reaction (PCR) approach with primers P1/P7 and R16F2n/R16R2. DNA from healthy leaves and sterile water was used as a negative control, while DNA from periwinkle infected with faba bean phyllody (16SrII-C), aster yellows (16SrI), tomato big bud (16SrII-D), sweet potato little leaf (16SrII-D), catharanthus phyllody (16SrVI), and sesame phyllody (16SrII-A) were used as positive controls and for restriction fragment length polymorphism (RFLP) comparisons. Nested 1.25-kb PCR products from infected plant samples were subjected to RFLP analysis with restriction endonucleases RsaI, AluI, HaeIII, HhaI, EcoRI, TaqI, Tru9I, and Sau3AI. The analysis showed that the alfalfa witches' broom phytoplasma (AWBP) belonged to the 16SrII group (peanut witches' broom) and that the AWBP was most similar to sweet potato little leaf (16SrII-D) but distinct from "Candidatus Phytoplasma aurantifolia," the cause of lime witches' broom in Oman. Other phytoplasmas infecting alfalfa have been reported from Europe and North America (1,3), but they belong to the 16SrVI (clover phyllody) and 16SrI (aster yellows) groups. An alfalfa witches' broom reported from Italy (2) forms a separate grouping (4). To our knowledge, this is the first report of a phytoplasma from the peanut witches' broom group infecting alfalfa in the Sultanate of Oman. References: (1) A. H. Khadhair et al. Microbiol. Res. 152:259, 1997. (2) C. Marcone et al. J. Plant Pathol. 79:211, 1997. (3) R. D. Peters et al. Plant Dis. 83:488, 1999. (4) E. Seemuller et al. J. Plant Pathol. 80:3, 1998.