Tomato Big Bud Research Articles

First report of Candidatus Phytoplasma australasia (16SrII- subgroup D) associated with virescence of Chia (Salvia hispanica L.) from India.

Chia (Salvia hispanica L., Lamiaceae) is an important commercial and medicinal crop recently popularized in India and widely cultivated in Karnataka (Joy et al., 2022). During the field survey of chia crop diseases, characteristic virescence like symptoms were observed at Main Agricultural Research Station, UAS, Raichur as well as at Mysuru and HD Kote region. The incidence was ranged from 2 - 4 per cent in an area of 30 hectares. Typical symptoms associated with chia are malformed shoot and/or inflorescence axis with reduced floral parts with greenish florets. The stem axis become thick, flattened, leaves are reduced towards terminal region. A total of five phytoplasma suspected samples and five suspected healthy samples were used for identification purpose. The Plant Genomic DNA Miniprep Kit (Sigma Aldrich, USA) was used to extract the DNA from five symptomatic and five asymptomatic samples and the DNA was used as template to amplify the phytoplasma-specific 16S rDNA gene using P1/P7 primers (Deng and Hiruki, 1991; Schneider et al., 1995) followed by nested PCR using R16F2n/R16R2 primers (Gundersen and Lee 1996). The expected 1.25-kb amplicon was detected from the suspected symptomatic samples. Nested PCR products were purified and sequenced from both the directions using ABIX370 Genetic Analyzer (Applied Biosystems, Waltham, MA). The analysis revealed that all five sequences shared 100 per cent identity with Candidatus Phytoplasma aurantifolia (OM649850, ON975012) and Tomato big bud phytoplasma (EF193359). The in-silico RFLP pattern of F2n/R2 primed region of 16S rDNA gene analyzed by using iPhyClassifier (Zhao et al. 2009) revealed that the sequence shared 98.72 per cent nucleotide sequence similarity with coefficient value of 1.00 to the reference strain RFLP pattern of 16Sr group II, subgroup D (witches'-broom disease of lime; U15442). Based on 16SrDNA sequences and in-silico RFLP analysis, the phytoplasma associated with the chia virescence was identified as a member of 16SrII-D group. Further, SecA gene was also amplified from the samples using SecAfor1/SecArev3 primer pair (Hodgetts et al., 2008). All samples produced ~400 bp products and sequenced as detailed above. Sequence analysis by nBLAST revealed 100 per cent similarity to Ca. P. australasia (MW020545) and Ca. P. aurantifolia isolate Idukki Kerala 1 (MK726369) both representing 16SrII-D group phytoplasma. The representative sequence (16Sr: PP359693, PP359694; secA:PP386558, PP386559) were deposited in GenBank. Chia virescence phytoplasma belonging to Ca. phytoplasma australasia has not been reported anywhere. The phytopathological studies associated with chia crop are very limited. Joy et al. (2022) reported the occurrence of foot rot disease caused by Athelia rolfsii. Several hosts are recorded to be associated with 16SrII D phytoplasma which includes china aster, eggplant and crotalaria (Mahadevakumar et al., 2017, Yadav et al., 2016a, b). Now the wide occurrence of the phytoplasma in the area might have transmitted by vectors. The occurrence of virescence is of great importance as it affects the overall yield which reduces the market value. To our knowledge, this is the first report of a group 16SrII-D phytoplasma associated with chia virescence in India.

History and Current Status of Phytoplasma Diseases in the Middle East.

Simple SummaryPhytoplasmas are microorganisms that have been reported to be associated with hundreds of plant diseases in most parts of the world. Several reviews were published regarding diseases associated with phytoplasmas in different countries. However, no comprehensive review is available on the phytoplasma diseases in the Middle East, which is an important region with arid to semi-arid conditions. This review describes the most common phytoplasmas that are associated with diseases in this part of the world. It also describes some of the insect vectors that help to transmit these phytoplasmas. Information is also presented regarding the distribution of the diseases and host ranges.Phytoplasmas that are associated with fruit crops, vegetables, cereal and oilseed crops, trees, ornamental, and weeds are increasing at an alarming rate in the Middle East. Up to now, fourteen 16Sr groups of phytoplasma have been identified in association with more than 164 plant species in this region. Peanut witches’ broom phytoplasma strains (16SrII) are the prevalent group, especially in the south of Iran and Gulf states, and have been found to be associated with 81 host plant species. In addition, phytoplasmas belonging to the 16SrVI, 16SrIX, and 16SrXII groups have been frequently reported from a wide range of crops. On the other hand, phytoplasmas belonging to 16SrIV, 16SrV, 16SrX, 16SrXI, 16SrXIV, and 16SrXXIX groups have limited geographical distribution and host range. Twenty-two insect vectors have been reported as putative phytoplasma vectors in the Middle East, of which Orosius albicinctus can transmit diverse phytoplasma strains. Almond witches’ broom, tomato big bud, lime witches’ broom, and alfalfa witches’ broom are known as the most destructive diseases. The review summarizes phytoplasma diseases in the Middle East, with specific emphasis on the occurrence, host range, and transmission of the most common phytoplasma groups.

Multilocus sequence analysis of Candidatus Phytoplasma aurantifolia associated with phyllody disease of gerbera from India.

Gerbera is the most popular cut flower known for its variety of colors and is grown across the world. Its production is challenged by numerous diseases affecting production and quality. During our survey, ten samples from the gerbera plants exhibiting phyllody disease symptoms were collected from Bangalore Rural District, Karnataka, India. The association of phytoplasma with the gerbera phyllody samples was confirmed by PCR using 16SrRNA, SecY, Ribosomal protein (rp) and SecA gene-specific primers. PCR products were amplified from all ten gerbera plants using phytoplasma-specific primers. The amplified PCR products were cloned and sequenced; the sequences of the ten clones were identical. Therefore, representative isolate (GePP1, Gerbera phyllody phytoplasma) was selected for further analysis. The sequence analysis showed that GePP1 shared maximum nucleotide (nt) identity of 97.1% (16SrRNA) with Eggplant big bud, 98.7 to 98.8% (SecY gene) with Tomato big bud, 99.2 to 99.6% (rp gene) with Alfalfa witches-broom (EF193371) and 99.1% (SecA gene) with Sesame phyllody phytoplasmas and that it belongs to the Ca. P. aurantifolia (16SrII) group. This result was well supported by the phylogenetic analysis showing GePP1 (16Sr RNA, SecY, rp and SecA genes) closely clustering with the Ca. P. aurantifolia 16SrII group isolates reported so far. The virtual RFLP pattern generated for the phytoplasma from gerbera was different (similarity coefficient 0.89) from the reference pattern of Ca. P. aurantifolia (16Sr II) subgroup after analysis with four enzymes (BfaI, Hha1, Sau3AI and RsaI). Based on the threshold similarity coefficient for a new subgroup (delineation should be set at 0.97), the GePP1 may be considered as new subgroup of Ca. P. aurantifolia (16SrII) group. This is the first report of Ca. P. aurantifolia belonging to 16Sr II group affecting gerbera in India. Keywords: Candidatus Phytoplasma aurantifolia; phyllody; gerbera; PCR; phylogenetic analysis.

Emerging incidence of tomato big bud disease associated with a 16srii-d phytoplasma in Andhra Pradesh and odisha states of India

Emerging incidence of tomato big bud disease associated with a 16srii-d phytoplasma in Andhra Pradesh and odisha states of India

Interaction between ‘Candidatus Phytoplasma australasiae’ and Tomato yellow leaf curl virus in tomato plants

To evaluate the effect of mixed infection of two main pathogens of tomato in Iran, the interaction between tomato big bud phytoplasma (TBBP, 16SrII-D) and Tomato yellow leaf curl virus (TYLCV) was investigated under greenhouse condition. Tomato plants were graft-inoculated either with TBBP or TYLCV alone or together simultaneously and non-simultaneously. Symptoms expression and incubation period of the diseases were evaluated on all inoculated plants. To assess pathogen development, the concentrations of TBBP and TYLCV were compared at 10, 20, 40 and 70 days post-inoculation using quantitative PCR assays. In all replications, the tomato plants doubly inoculated with TBBP + TYLCV showed milder symptoms and longer incubation period than those singly inoculated with TBBP or TYLCV. Results also showed a reduction of TYLCV and TBBP concentrations in doubly inoculated plants when compared to single infection, although these reductions were not always statistically significant. In the tomato plants simultaneously inoculated with TBBP and TYLCV, the phytoplasma and virus concentrations were significantly lower at 70 days post-inoculation. These results suggest an antagonism between TYLCV and TBBP when infecting the tomato plant.

Detection of phytoplasma causing tomato big bud disease in Syria

Detection of phytoplasma causing tomato big bud disease in Syria

Increasing incidence of tomato big bud phytoplasma in Ranga Reddy District of Telangana State, India

Tomato is an important vegetable crop and India ranks second in area and production of tomato worldwide. During field surveys in Moinabad and Shamshabad Mandals in Ranga Reddy district of Telangana from 2014–2016, symptoms like big bud and phyllody were observed on tomato. The affected plants did not produce any fruit. The phytoplasma strains were detected and characterized using universal and nested primer pairs of phytoplasma with amplification of 16S rRNA and secA genes. BLAST analysis of 1.25 kb 16S rDNA partial sequences of nested PCR products and 880 bp of secA gene products obtained from symptomatic TBB (tomato big bud) samples revealed 99% sequence identity with strains of ‘Ca. Phytoplasma australasia’ (16Sr II group). Phylogenetic analysis and virtual RFLP analysis of 16SrDNA sequences of tomato big bud phytoplasma (TBBP) strain also suggested the closest relationship with ‘Ca. P. australasia’16Sr II-D subgroup related strain. Present study confirmed association of 16Sr II-D subgroup of phytoplasma associated with tomato big bud disease in Telangana State of India.

Occurrence, identification and transmission of the phytoplasma associated with tomato big bud disease and identification of its vector and weed host in Pakistan

Tomato (Solanum lycopersicum L.) plants showing stunting, big bud, leaves yellowing or reddening and witches’-broom symptoms were observed since 2009 in Pakistan. A weed Parthenium hysterophorus grown in and around tomato fields also exhibited witches’-broom like symptoms. Fluorescence light microscopy of hand-cut stem stalk sections treated with Dienes’ stain showed blue areas in the phloem region of both tomato and P. hysterophorus symptomatic plants that indicated the association of phytoplasma with the complex. Amplification of 1.2 kb 16S rDNA fragment in nested PCR confirmed that the symptomatic tomato and P. hysterophorus plants are infected by a phytoplasma. Partial sequencing of 16S rRNA (GenBank accession: LT671581 and LT671583) and virtual restriction fragment length polymorphism confirmed that the phytoplasma associated with both plant species had the greatest homology to 16SrII-D subgroup. Disease was successfully transmitted by grafting and leafhopper Orosius albicinctus in tomato plants. This is the first report of natural occurrence of 16SrII-D phytoplasma in tomatoes and a weed P. hysterophorus in Pakistan.

Detection, Characterization and In-Silico Analysis of Candidatus Phytoplasma australasiae Associated with Big Bud Disease of Tomato in India

Tomato plants showing witches broom symptoms were collected from different states of India. The presence of phytoplasma infection was confirmed by PCR using phytoplasma-specific primer of 16S rRNA and SecY gene. The sequence analysis of 16S rRNA and SecY gene of eight tomato big bud phytoplasmas showed maximum nucleotide (nt) identity of 95–100% with Peanut WB group (16SrII). Further in-silico RFLP analysis of 16S rRNA gene of TBB-Pun1, TBB-Ban, TBB-mal, TBB-Guj and TBB-Vns showed similarity coefficient of 0.68–0.95. Since threshold similarity coefficient for classifying the phytoplasma into new subgroup is set at 0.97, the strain under study significantly distinct from the representative strains in the subgroups of pea nut witches broom. Further, the phylogenetic analysis of tomato big bud phytoplasmas revealed that, they are closely clustered with peanut witches’-broom strains (16Sr II), specifically within the 16Sr II-D and 16Sr II-A subgroups. A comprehensive recombination analysis showed the evidence of both intra and inter-species recombination in seven tomato big bud isolates with most part of their 16Sr RNA F2nR2 fragments descending from Ca.P.brasiliense (16Sr XV) as major parent, except isolate TBB-Vns which had an intra species recombination with Cactus witches-broom-16Sr II-L as major parent. Similarly, in case of SecY gene, all the seven isolates have intra-species recombination with major portion descending from Vinca virescence-[16Sr VI-A] and Potato purple top wilt-[16Sr XVIII-B]. The genetic similarities and the potential threat of this new phytoplasma belonging to 16Sr II group of Peanut witches’ broom’ group infecting tomato in India are discussed.

THE FIRST REPORT OF A 16SrXII-A PHYTOPLASMA ASSOCIATED WITH TOMATO BIG BUD DISEASE IN IRAN

In 2013 surveys, up to 7.5% incidence of big bud disease was observed in tomato fields of Kazerun area (Fars province, Iran). The main disease symptoms were big bud, little leaf, flower virescence, phyllody, proliferation and sterility. To investigate the phytoplasma presence, total DNAs extracted from four symptomatic and four symptomless tomato plants were tested by nested PCR using P1/P7 primer pair followed by R16F2n/R16R2 primers (Lee et al., 1998). Amplicons of ca. 1.8 and 1.2 kb, respectively were amplified in samples of symptomatic plants but not of symptomless ones. Four P1/P7 amplicons from symptomatic tomato plants were cloned and sequenced. The obtained 16S rDNA sequences showed 100% sequence identity with each other and a representative of these sequences deposited in GenBank (Accession No. KX098490). BLAST search using full length 16S rRNA gene sequence revealed that Kazerun tomato big bud (KTBB) sequence showed 100% identity with a 'Candidatus Phytoplasma solani' strain (AF248959), representative of 16SrXII-A subgroup. Computer-simulated restriction analysis using iPhyClassifier showed that the RFLP profile of KTBB 16S rDNA F2nR2 fragment was identical (similarity coefficient 1.00) to the reference pattern of 16SrXII-A (AF248959). Phylogenetic analysis revealed that KTBB phytoplasma clustered with16SrXII group phytoplasmas closest to 16SrXII-A subgroup reference strain (AF248959). To our knowledge this is the first report of a 16SrXII-A phytoplasma associated with TBB disease in Iran. 16SrXII- related phytoplasmas have been previously reported from grapevine (Salehi et al., 2014) and Cannabis sativa (Vali Sichani et al., 2011) in Iran.

First Report of Tomato Big Bud Caused by a 16SrII-C Phytoplasma in Tanzania

First Report of Tomato Big Bud Caused by a 16SrII-C Phytoplasma in Tanzania

Characterisation of a phytoplasma associated with Petunia witches' broom disease in Iran

Petunia hybrida (Solanaceae) is an economically important ornamental plant species that is used in residential and commercial landscapes. Varieties are diverse and available in a range of colours (Abu-Qaoud et al, 1; Hiuts et al, 3). In July 2012, symptoms of witches' broom, yellowing, little leaf, phyllody and virescence were observed in Petunia in Sistan and Baluchestan province, Iran (Fig. 1a, b). Total DNA was extracted separately from 0.5 g of shoot material of two symptom-bearing and one symptomless plant (Fig. 1c) using a modified cetyltrimethylammonium bromide (CTAB) method (Doyle & Doyle, 2). The samples were analysed for detection of phytoplasma DNA by both direct and nested PCR assays using phytoplasma universal 16S rDNA primer pairs P1/P7 and R16F2n/R16R2 that amplify fragments of 1800 bp and 1250 bp, respectively (Lee et al., 4). The 1800 and 1250 bp PCR products were obtained following direct and nested PCR from all symptom-bearing but not from symptomless plants. The 1250 bp PCR product was directly sequenced. BLAST analysis of the partial 16S rDNA sequence (GenBank Accession No. JX570935) revealed that the phytoplasma associated with Petunia witches' broom (PtWB) shared 100% identity with some phytoplasmas related to 16Sr-II phylogenetic group, ‘Candidatus Phytoplasma aurantifolia’, such as chickpea phyllody phytoplasma (FJ870549.1), tomato big bud phytoplasma (JQ868448), ‘Ca. P. aurantifolia’ (JX104335) and Echinacea witches' broom phytoplasma (JF340075.1). This result was further confirmed by the analysis using iPhyClassifier online tool (http://www.plantpathology.ba.ars.usda.gov/cgibin/resource/iphyclassifier.cgi) where it was determined that PtWB phytoplasma is related to 16Sr group-II, subgroup D. The phylogenetic neighbour-joining tree (MEGA 5 software) based on the partial 16S rDNA sequences (1250 bp) of the PtWB phytoplasma and several reference phytoplasmas corroborated these results and showed that PtWB phytoplasma is closely related to ‘Candidatus Phytoplasma australasiae’ in the 16Sr-II phytoplasma clade (Fig. 2). Salehi et al. (unpublished) have recently detected a phytoplasma associated with phyllody symptoms in Petunia by PCR in Fars province, Iran. However, this is the first report of molecular characterisation of a phytoplasma associated with PtWB disease in Iran.

Association of pigeon pea witches’ broom phytoplasma infectingCarpobrotus edulis(L.) N.E. Br in India

Leaf yellowing and little leaf symptoms with reduced size of flowers were observed in Carpobrotus edulis at Gorakhpur, Uttar Pradesh, India in the years 2012–2013. Phytoplasma presence was confirmed in symptomatic C. edulis leaves using phytoplasma universal primers P1/P7 and R16F2n/R16R2 that produced expected size amplicons of ~1.8 kb and ~1.2kb, respectively. BLAST analysis of 16S rDNA sequence of the phytoplasma detected revealed 99% similarity with those of tomato big bud, Brassica rapa phyllody, Iranian almond witches’ broom, Phlox drummondii, sesame phyllody and Iran eggplant big bud all belonging to 16SrIX group. Phylogenetic analysis of the C. edulis phytoplasma 16S rDNA sequence confirmed its closest relationship with members of the 16SrIX group. Virtual RFLP analysis of 1.25 kb sequence of 16S rDNA identified it as a new strain in the 16SrIX-C subgroup that is for the first time reported in C. edulis species worldwide.

Molecular Detection and Identification of a 16SrVI Group Phytoplasma Associated with Tomato Big Bud Disease in Xinjiang, China

AbstractIn 2010, tomato plants with big bud symptoms were observed in Xinjiang, China. PCR products of approximately 1.2 and 2.8 kb were amplified from infected tomato tissues but not from asymptomatic plants. A comparison of 16S rDNA sequences showed that the casual tomato big bud (TBB) phytoplasma was closely (99%) related to the ‘Candidatus Phytoplasma trifolii’ (16SrVI group). The TBB phytoplasma clustered into one branch with the Loofah witches'‐broom phytoplasma according to the 23S rDNA analysis but with no other member of the 16SrVI group. The cause of TBB symptoms was identified as ‘Ca. Phytoplasma trifolii' (16SrVI group) by PCR, virtual RFLP and sequencing analyses. This is the first report of a phytoplasma related to ‘Ca. Phytoplasma trifolii' causing TBB disease in China.

First Report of a 16SrII-D Subgroup Phytoplasma Associated with Pale Purple Coneflower Witches'-Broom Disease in Australia.

Pale purple coneflower, Echinacea pallida (Nutt.) Nutt., is an herbaceous perennial cultivated for its ornamental and medicinal properties. In 2005, phytoplasma-like symptoms, including virescence, phyllody, and chlorotic leaves, were first observed in coneflower fields in northern Tasmania, Australia. During the 2010-2011 growing season, the incidence of affected plants was estimated to be 12% within a single field. Total DNA was extracted from symptomatic plants with a DNeasy Plant Mini Kit (QIAGEN Inc., Valencia, CA) according to the manufacturer's instructions. DNA was also extracted, as described above, from asymptomatic coneflower seedlings obtained by germinating surface-sterilized seed on water agar. DNA was amplified via a nested PCR using universal primer pairs P1/P7 followed by R16F2n/R16R2 to detect putative phytoplasmas (2). Amplifications yielded expected products of 1.8 and 1.2 kb, respectively, only from symptomatic samples. Subsequently, PCR products from six arbitrarily selected samples were used for sequencing (Genome Lab Dye Terminator Cycling Sequence with Quick Start Chemistry) and read in a CEQ8000 sequencer (Beckman Coulter Inc., Brea, CA). Sequence homology indicated 100% similarity between isolates designated EWB1 to EWB4 (GenBank Accession Nos. JF340075 and JF340077 to JF340079) and between EWB5 and EWB6 (JF340076 and JF40080). Sequence homology between the two observed groups was 99.7%, resulting from a 4-bp difference in the R16F2n primer region. Blast search revealed isolates EWB1 to EWB4 were 100% homologous with Catharanthus roseus phytoplasma (EU096500.1), Tomato big bud phytoplasma (EF193359.1), Scaevola witches'-broom phytoplasma (AB257291.1), and Mollicutes sp. (Y10097.1 and Y10096.1). Moreover, isolates EWB5 and EWB6 shared 99% sequence identity with the above isolates. iPhyClassifier (4) was used to perform sequence similarity and generate virtual restriction fragment length polymorphism (RFLP) profiles. The 16S rDNA sequence of isolates EWB1 to EWB4 and EWB5 to EWB6 shared 100 and 99.7% similarity, respectively, to the 'Candidatus Phytoplasma australasiae' reference strain (Y10097). RFLP profiles from all isolates suggested that they belonged to the 16SrII-D subgroup. To our knowledge, this is the first report of a 16SrII-D subgroup phytoplasma infecting E. pallida in Australia. Aster yellow phytoplasmas (16SrI-C subgroup) infections of E. purpurea have been recorded in Slovenia (3) and southern Bohemia (1).

Brote Grande, A New Phytoplasma Associated Disease of Chile Peppers in the Desert Southwest

Chile is one of the most important crops in New Mexico, contributing both to the agricultural economy and cultural identity of the state. Diseases are a major constraint on chile production in New Mexico and across the Desert Southwest. Chile producers in New Mexico recently reported a disorder of unknown etiology that was observed in increasing frequency for the past four years. Affected plants have a bushy appearance, develop overly large green calyces instead of normal flowers, and fail to set fruit. This characteristic phyllody has led the disorder to be referred to as “brote grande” which is Spanish for “big bud.” The phyllody is reminiscent of the aberrant flower development associated with tomato big bud, a phytoplasma disease of tomatoes. Microscopic analysis, including light microscopy and TEM along with PCR detection using phytoplasma specific primer sets, indicates that brote grande disease is associated with a novel phytoplasma. Field surveys conducted in 2008 and 2009 indicate that this new disease is widely distributed at low levels across chile production areas in New Mexico and Arizona. Accepted for publication 10 January 2011. Published 1 March 2011.

Brote Grande, a New Phytoplasma-Associated Disease of Chile Peppers.

Chile is one of the most important crops in New Mexico, contributing both to the agricultural economy and cultural identity of the state. Chile producers in New Mexico and Arizona have reported a disorder of unknown etiology that has increased in frequency for the past several years. Affected plants have a bushy appearance, develop overly large green calyces instead of normal flowers, and fail to set fruit. This characteristic phyllody is similar to symptoms associated with other phytoplasma-caused diseases, such as tomato big bud, and has led chile producers to refer to the disorder as "brote grande", which is Spanish for "big bud". PCR analysis using the phytoplasma-specific primer pairs P1/Tint and P1/P7 (4) produced amplicons of the expected size (~1.6 kb) from symptomatic but not healthy samples. Direct sequencing of the P1/P7 PCR amplicons determined that they contained the expected 16S rRNA and internal transcribed spacer (ITS) sequences and included the tRNAIle typically found in phytoplasma ITS regions. BLAST analysis of the brote grande sequence (GenBank Accession No. FJ525437) indicated it is most closely related (99% identity) to sequences reported for previously characterized 16Sr group VI phytoplasmas, such as 'Candidatus Phytoplasma trifolii' (Accession No. AY390261) and the Vinca virescence (Accession No. AY500817) phytoplasma. 'Candidatus phytoplasma trifolii' is synonymous with beet leafhopper virescence, which was reported as a cause of tomato big bud in California during the mid 1990s (3). The brote grande phytoplasma was less related to other phytoplasmas known to affect peppers such as the 16Sr group XII stolbur of pepper phytoplasma (Accession No. AF248959) and newly described 16Sr group I phytoplasmas described in peppers in Cuba (Accession No. DQ286947) and Mexico (Accession No. DQ092321) (1,2). The brote grande phytoplasma is also distinct from other phytoplasmas, such as potato purple top and tomato little leaf that are common in Mexico, affecting solanaceous crops in the region (2). Although the disease frequency never exceeded 5% in any given field, plants displaying brote grande symptoms were observed in the majority of chile pepper fields examined from July to September of 2008. The presence of the brote grande associated phytoplasma was confirmed by PCR and sequence analysis of symptomatic plants from 10 different fields ranging from Las Cruces, NM to Tucson, AZ, indicating that brote grande disease is widespread across the major chile-producing areas of the Desert Southwest. The brote grande phytoplasma sequence was the only phytoplasma sequence detected in any of the symptomatic chile samples. Taken together, the etiology, PCR, and DNA sequence results all indicate that brote grande of chile is a new disease of chile peppers associated with infection by a novel 16Sr group VI phytoplasma and that this disease is distributed across the major chile-producing areas of the Desert Southwest.

Anti‐metabolic effects of Galanthus nivalis agglutinin and wheat germ agglutinin on nymphal stages of the common brown leafhopper using a novel artificial diet system

The common brown leafhopper, Orosius orientalis (Matsumura) (Homoptera: Cicadellidae), previously described as Orosius argentatus (Evans), is an important vector of several viruses and phytoplasmas worldwide. In Australia, phytoplasmas vectored by O. orientalis cause a range of economically important diseases, including legume little leaf (Hutton & Grylls, 1956), tomato big bud (Osmelak, 1986), lucerne witches broom (Helson, 1951), potato purple top wilt (Harding & Teakle, 1985), and Australian lucerne yellows (Pilkington et al., 2004). Orosius orientalis also transmits Tobacco yellow dwarf virus (TYDV; genus Mastrevirus, family Geminiviridae) to beans, causing bean summer death disease (Ballantyne, 1968), and to tobacco, causing tobacco yellow dwarf disease (Hill, 1937, 1941). TYDV has only been recorded in Australia to date. Both diseases result in significant production and quality losses (Ballantyne, 1968; Thomas, 1979; Moran & Rodoni, 1999). Although direct damage caused by leafhopper feeding has been observed, it is relatively minor compared to the losses resulting from disease (P Tr E bicki, unpubl.).

An Application of Space-Time Analysis to Improve the Epidemiological Understanding of the Papaya-Papaya Yellow Crinkle Pathosystem

Yellow crinkle disease of papaya is a serious threat to papaya production in Australia. Space-time point pattern analysis was used to study the spatial and temporal dependence of two phytoplasma strains that cause yellow crinkle: tomato big bud (TBB) and sweet potato little leaf V4 (SPLL-V4). Incidence data for both phytoplasma strains were obtained from a field study conducted in Katherine, NT, Australia, between January 1996 and May 1999. The primary ecological and epidemiological question of interest was to elucidate the scale of spatial or spatio-temporal aggregation of phytoplasma-infected papaya plants. The hypothesis was that there would be a contagion process, where TBB- and SPLL-V4-infected papaya would be aggregated and not random. To test this hypothesis, a point pattern spatial analysis using Monte Carlo simulation was initially applied to the incidence data. Results of this analysis suggested that SPLL-V4 infected papaya plants displayed aggregation with spatial dependence up to 30 m (10 to 15 plants along or across rows), whereas there was not strong evidence to suggest that TBB-infected papaya plants were aggregated. However, when a space-time point pattern analysis was subsequently used to simultaneously test for the interaction between space and time, there was strong evidence (P < 0.01 for SPLL-V4 and P < 0.10 for TBB) to suggest a space-time interaction for both SPLL-V4 and TBB. For SPLL-V4, a space-time risk window of approximately 10 months and 20 m was detected, whereas for TBB, this risk window was 5 months and 10 m. The results of these studies support the hypothesis that papaya infection by both phytoplasma strains appears to be the result of a contagion process, providing support for the contention that insect vectors are the most likely mechanism for acquisition, dispersal, and transmission. Accepted for publication 26 April 2007. Published 26 July 2007.

First Report of a "Candidatus Phytoplasma australiense"-Related Strain in Lucerne (Medicago sativa) in Australia.

Australian lucerne yellows (ALuY), a phytoplasma-associated disease, is a major problem in Australia that causes the pasture seed industry millions of dollars in losses annually (3). Samples were collected from lucerne (Medicago sativa L.) plants exhibiting symptoms indicative of ALuY (4) in a seed lucerne paddock (cv CW 5558) at Griffith, southwestern New South Wales (NSW), Australia, in November 2005 and again in January 2006. Samples were kept at 4°C and processed within 36 h of collection. Total DNA was extracted from approximately 0.3 g of leaf midribs and petioles of each plant sample and used as template in a nested PCR assay with phytoplasma universal primer pairs P1/P7 and fU5/m23sr. PCR products resulting from the first amplification were diluted (1:30) with sterile distilled water (SDW) before reamplification with fU5/m23sr. DNA of Australian tomato big bud (TBB) phytoplasma and SDW were used as positive and negative assay controls, respectively. Ten of fifteen plant samples collected in November tested positive for phytoplasma DNA. Restriction digestion profiles of nested PCR amplicons with HpaII endonuclease were the same for all symptomatic plants but differed from the control. Phytoplasma identity was determined by sequencing two nested PCR products that yielded identical sequences. One was deposited in the GenBank database (Accession No. DQ786394). BLAST analysis of the latter sequence revealed a >99.6% similarity with "Candidatus Phytoplasma australiense" (L76865) and related strains papaya dieback (Y10095), phormium yellow leaf (U43570), strawberry green petal (AJ243044), and strawberry lethal yellows (AJ243045). Direct PCR with primers FP 5'-GCATGTCGCGGTGAATAC-3' and RY 5'-TGAGCTATAGGCCCTTAATC-3' designed to specifically amplify DNA of "Ca. P. australiense" detected the phytoplasma in 8 of 40 samples collected in January. Whether this phytoplasma is the etiological agent solely responsible for ALuY is currently under investigation. "Ca. P. asteris" and stolbur group (16SrXII) phytoplasmas have been reported in lucerne in the United States (2) and Italy (1), respectively. Within the stolbur group 16SrXII, "Ca. P. australiense" and stolbur phytoplasma are regarded as separate species and both are distinct from "Ca. P. asteris", a group 16SrI strain. To our knowledge, this is the first report of a "Ca. P. australiense" related strain in lucerne.