Papaya Yellow Crinkle Research Articles

First report of a 'Candidatus Phytoplasma aurantifolia'-related strain associated with faba bean phyllody symptoms in India.

Vicia faba L. commonly known as broad bean or faba bean is one of the most widely grown protein rich legume crops. Out of more than 50 faba bean-producing countries, about 90% production is concentrated in the Asian, European Union (EU), and African region (FAO, 2020). Owing to its high nutritional value, both the fresh pods and dry seeds are consumed. During March 2022, some plants with little leaf and phyllody symptoms such as leaf-like floral structures were observed in the experimental fields of Indian Agricultural Research Institute (IARI), New Delhi (Fig. 1 a, b, c). The twig samples were collected from two individual symptomatic and one from asymptomatic plant. DNA was extracted using CTAB (cetyl trimethyl ammonium bromide) method (Ahrens and Seemüller, 1992; Marzachi et al. 1998) and examined for the association of phytoplasma through nested PCR using the universal primers P1/P7 and R16F2n/R16R2 targeting the 16SrRNA gene (Deng and Hiruki 1991; Gundersen and Lee 1996) and the other set of primers secAfor1/secArev3 and secAfor2/secArev3 targeting secA gene (Hodgetts et al. 2008). The DNA from symptomatic plants resulted the amplicons of 1200bp and 840bp specific to 16S rRNA and secA gene respectively. The gel purified PCR products were cloned into pGEM®-T Easy Vector system (Promega) and outsourced for Sanger sequencing at Agri Genome Labs, Kerala, India. The resultant 16S rRNA sequences (GenBank Acc. No. OP978231, OP978232) and secA sequences (ON715392 and ON715393) were examined through NCBI BLASTn analysis. The 16S rRNA sequences of the V. faba strains shared a minimum of 99.85% similarity with the phytoplasma strain causing little leaf and phyllody disease of sesame in India (MW622017) and a maximum of 100% sequence identity with the Vigna radiata phyllody and necrosis phytoplasma strain of Jodhpur (OP935760) India, whereas the secA gene sequences showed 100% identity with Tephrosia purpurea witches'-broom phytoplasma (MW603929) from China and a minimum of 91.14% similarity with 'Candidatus Phytoplasma aurantifolia' (MW020541) from India. The pairwise comparison results were completely in support of the corresponding phylogenetic sequence analysis results of 16SrRNA and secA gene sequences of faba bean strains in comparison with other strains retrieved from GenBank database, wherein the faba bean strains got clustered with 16SrII-D subgroup related strains (Fig. 2 a and b). Virtual RFLP analysis through iPhyClassifer tool through in silico digestion of R16F2n/R2 region of 16S rRNA gene of the faba bean strain using 17 restriction endonuclease enzymes resulted in the RFLP profiles similar to that of the profile of phytoplasma subgroup 16SrII-D (Y10097: papaya yellow crinkle) used as reference strain with a similarity coefficient value of 1.0. All the results of this investigation confirmed the association of 'Candidatus phytoplasma aurantifolia' (16SrII-D) with the diseased faba bean plants in this study. Previous reports of phytoplasma infecting faba bean include a group 16SrIII strain detected in Spain in 2004 (Castro and Romero), a subgroup 16SrII-D strain detected in Sudan in 2012 (Alfaro-Fernandez et al.), a group 16SrII strain detected in Saudi Arabia in 2014 (Al-Saleh and Amer), and subgroup 16SrIII-J strains detected in Egypt in 2014 (Hamed et al.) and in Peru in 2021 (Torres-Suarez et al.). To the best of our knowledge, these findings, document the first report of the association of 'Candidatus Phytoplasma aurantifolia' (subgroup 16SrII-D) with faba bean plants in India. This report necessitates further research on the status of distribution of this phytoplasma strain in other locations and hosts in the country so as to develop possible strategies to contain its further spread and management of the disease.

First Report of a ‘Candidatus Phytoplasma aurantifolia’ Strain Associated with Virescence, Floral Proliferation, and Dwarf Symptoms on Indigofera suffruticosa in Taiwan

Indigofera suffruticosa Mill., generally known as anil or Guatemalan indigo, is a perennial shrub planting in tropical and subtropical regions, including Taiwan; it was commonly used to produce the indigo dye, which is still being used in many personal workshops in Taiwan. There were a few reported cases of the association of phytoplasma with Indigofera spp. In 1983, I. suffruticosa showing phyllody and small leaf symptoms was first described, but no molecular identification was conducted (Albanese et al. 1995). Indigofera linifolia with little leaf, phyllody, and proliferation symptoms and Indigofera colutea showing little leaf symptoms in Australia caused by phytoplasma strains were reportedly associated with papaya yellow crinkle disease (Padovan and Gibb 2001). Affected Indigofera juncea and Indigofera hirsuta exhibiting witches’-broom symptoms were reported to be the same as the sesame phyllody phytoplasma isolate in Thailand (Nakashima et al. 1995). In February 2016, I. suffruticosa plants showing virescence, floral proliferation, and dwarf symptoms were collected from commercial fields of Mingjian Township, Nantou County, Taiwan. Two each of symptomatic and asymptomatic samples were collected from the field for further study. Transmission electron microscopic observation revealed the presence of the pleomorphic phytoplasma cells only in the phloem sieve cells of the two symptomatic I. suffruticosa plants. The two asymptomatic and two symptomatic samples were used for total DNA extraction with the modified CTAB method, and the amplicons of the nested PCR with universal primer pairs for nearly full-length 16S rDNA amplification (P1/P7 followed by R16F2n/R16R2) were electrophoresed in 0.8% agarose gel (Fulton et al. 1995; Lee et al. 1998). A 1.2-kb fragment (GenBank accession no. KX268346) was identified only from the two symptomatic I. suffruticosa plants. Analysis of 16S rDNA sequences by iPhyClassifier indicated that the phytoplasma associated with virescence symptoms in I. suffruticosa shared a 98.2% identity with that of the ‘Candidatus Phytoplasma aurantifolia’ reference strain (GenBank accession no. U15442). Virtual restriction fragment length polymorphism patterns were identical to those of the 16SrII-V subgroup (Zhao et al. 2009). Taken together, the results indicated that the phytoplasma associated with I. suffruticosa in Taiwan is a ‘Ca. P. aurantifolia’-related strain. The occurrence of phytoplasma in I. suffruticosa could have direct implication on economic profit for growers and the handicraft industry in Taiwan. To our knowledge, this is the first report of a 16SrII-V subgroup phytoplasma causing virescence, floral proliferation, and dwarf symptoms on I. suffruticosa in Taiwan.

First report of a 16SrII phytoplasma associated with a witches' broom disease of Tamarix aphylla in Iran

Tamarisk (Tamarix aphylla) has been used for windbreaks near agricultural crops and as shade trees in rural desert margins in Iran. Symptoms associated with tamarisk witches' broom (TWB) disease, including proliferation of shoots with short internodes and little leaf symptoms (Figs. 1-2) were observed in 34% of trees surveyed in an area of 3500 ha in Chah Afzal (Yazd province, Iran) during 2014-15. Nested polymerase chain reaction using the P1/P7 primers (Deng & Hiruki, 1; Schneider et al., 5) followed by the R16mF2/R16mR2 and R16F2n/R16R2 primer pairs (Gundersen & Lee, 3) was carried out on DNA extracted from six diseased and three symptomless tamarisk plants. After 35 cycles, DNA fragments of c. 1.8, 1.4 and 1.25 kb respectively were obtained from each of the diseased tamarisk samples but not from symptomless samples. Restriction fragment length polymorphism (RFLP) analysis of R16F2n/R16R2 amplicons using AluI, HaeIII, HhaI, RsaI, MseI and TaqI restriction enzymes showed profiles identical to those of phytoplasmas belonging to the16SrII group. R16F2n/R16R2 sequences from six samples were identical to each other and a representative sequences was deposited in GenBank (Accession No. MF427724). BLAST analysis showed 99% nucleotide identity with other 16SrII-D phytoplasmas including parsley witches' broom phytoplasma (KU501295), which is also found in Iran. Phylogenetic analysis using the neighbour-joining method (MEGA7) showed that the TWB phytoplasma clustered within the 16SrII group closest to papaya yellow crinkle (PYC) phytoplasma (Y10097) which is the type strain of the 16SrII-D group. Computer-simulated analysis with seventeen restriction endonucleases using iPhyClassifier (Zhao et al., 6) showed that the RFLP pattern derived from the TWB phytoplasma 16S rRNA gene was different from the reference patterns of all previously established 16SrII subgroups. The most similar was the RFLP pattern of PYC phytoplasma (Y10097) with a similarity coefficient of 0.97. This is the first report of a 16SrII phytoplasma associated with TWB disease in Iran and worldwide. TWB disease occurs near field crops heavily infected with 16SrII phytoplasmas such as alfalfa witches' broom (Esmailzadeh Hosseini et al., 2) and sesame phyllody (Salehi et al., 4) in Yazd and therefore tamarisk may have an important role in the epidemiology of the disease.

Characterization of 16SrII-D subgroup associated phytoplasmas in new host plants in Egypt

Samples of three plant species displaying phytoplasma symptoms were collected from Kafrelsheikh and Al-Gharbia governorates during 2014. Witches’ broom and virescence symptoms were observed in periwinkle (Catharanthus roseus). Onion (Allium cepa) plants showed yellowing, streaks and twisting and Opuntia abjecta with proliferation and cylindrical of cladodes. Total DNA was extracted from symptomatic and asymptomatic plants, and phytoplasma were detected in all 12 symptomatic plants collected through direct and nested PCR assays with primers P1/P7 and R16F2n/R16R2. The results of phylogenetic analysis revealed that the phytoplasma isolates belong to 16SrII group. With a nucleotide identity greater than 98.7% with three members of 16SrII group, Papaya yellow crinkle, Y10097; “Ca. P. aurantifolia”, U15442; and peanut witches’ broom, Al33765, the strains identified in this study are “Ca. P. aurantifolia”-related strains. Virtual RFLP analysis of the 16S rRNA gene sequences with 17 restriction enzymes confirmed that the phytoplasma isolates belong to the “Candidatus Phytoplasma australasia” 16SrII-D subgroup. To the best of our knowledge, periwinkle, onion and Opuntia abjecta are considered new hosts for 16SrII group in Egypt.

Detection and molecular characterization of phytoplasmas associated with vegetable and alfalfa crops in Qassim region

ABSTRACTDuring a survey conducted in Qassim province, Saudi Arabia, in the year in 2015, 120 samples of carrot (Daucus carota subsp. sativus), onion (Allium cepa), faba bean (Vicia faba), green mustard (Brassica juncea) and alfalfa (Medicago sativa) plants displaying symptoms reminiscent of phytoplasma diseases were collected and tested for phytoplasma infection. Phytoplasma-specific PCR products were only amplified from symptomatic plants by nested-PCR. Disease incidence ranged from 3.14% in alfalfa crop fields 1 year after cultivation to 77.48% in 3-year-old fields. In the five carrot fields sampled in this study, the incidence changed from 3.2% to 100% after 7 months of cultivation. Phylogenetic analysis revealed that all Qassim phytoplasma isolates belong to the 16SrII group. Most of them shared 100% identity with papaya yellow crinkle (16SrII-D Y10097). The results from phylogenetic and virtual restriction fragment length polymorphism analyses of the 16S rRNA gene sequence confirmed that the phytoplasma of Qassim isolates under study is a member of 16SrII-D subgroup. To the best of my knowledge, the onion and green mustard are considered new hosts for the 16SrII group; therefore, this is the first report on the association of phytoplasma with diseases of faba bean, onion, carrot, mustard and alfalfa in Qassim province, Saudi Arabia.

First report of a 16SrII‐D phytoplasma ‘Candidatus Phytoplasma australasia’ associated with a tomato disease in India

Tomato (Solanum lycopersicum) is an important cash crop in India. Recently, symptoms of leaf yellowing and curling, little leaf, severe stunting and phyllody (Fig. 1) were observed in tomato fields in the Meerut district of western Uttar Pradesh with incidence of 5-8%. To investigate whether or not a phytoplasma is associated with the disease, eight tomato plants with indicative symptoms and eight without were collected from two fields belonging to Samaspur-Surani village, Meerut. Total DNA was extracted from midribs (DNeasy Plant Mini Kit, Qiagen, Germany) and used as template in a nested PCR assay to amplify the phytoplasma 16S rRNA gene using universal phytoplasma primers P1/P7 (Deng & Hiruki, 1991) and R16F2/R16R2 (Gundersen & Lee, 1996). Bands of expected size (1.8 and 1.26 kb, respectively) were produced in all samples from symptom-bearing plants, but not for the symptomless ones (Fig. 2). Three PCR products amplified with P1/P7 samples were selected and purified (QIAquick Gel Extraction kit, Qiagen, Germany) and directly sequenced. The resultant 16S rDNA sequences showed 100% of sequence identity with each other, and the consensus sequence was deposited in GenBank (Accession No. JX104335). BLAST analysis showed the highest sequence identity (100%) with that of members of group 16SrII ‘Candidatus Phytoplasma aurantifolia’ including the Papaya yellow crinkle phytoplasma strain Y10097 designated as ‘Ca. Phytoplasma australasia’ that belongs to subgroup 16SrII-D. Phylogenetic analysis supported BLAST comparisons since the Meerut tomato phytoplasma (Tomato-TMT) clustered in a single distinct branch that embraces ‘Ca. Phytoplasma australasia’ (Fig. 3). iPhyClassifier (Zhao et al., 2009) was used to perform sequence similarity and generate virtual restriction fragment length polymorphism (RFLP) profiles. The 16S rDNA sequence of Tomato-TMT isolates exhibited RFLP profiles identical to those of the reference strain Y10097, ‘Ca. P. australasia’, subgroup 16SrII-D (Fig. 4). To our knowledge, this is the first report of a 16SrII-D phytoplasma in India affecting the tomato crop. The results have significant phytosanitary impact for the epidemiology of 16SrII phytoplasma diseases in the tomato industry of Uttar Pradesh, India. This work was supported by Centre of Excellence in Agriculture Biotechnology, Council of Science & Technology, Uttar Pradesh and Bioinformatics Infrastructure Facility funded by DBT, Government of India.

First report of a 16SrII group phytoplasma associated with die‐back diseases of papaya and citrus in Ethiopia

Citrus orange (Citrus aurantifolia) and papaya (Carica papaya) are amongst the most common fruits grown in Ethiopia and the area devoted to their cultivation has increased considerably in the last six decades (Seifu, 2003). Recently decline disorders of both citrus and papaya have been reported as causing significant yield losses amongst the major farms growing these crops in the country. Symptoms in citrus include leaf interveinal chlorosis, mosaic or mottling, a reduction in the size and curling of leaves. In severe cases, premature fruit fall, twig drying and die-back, and death of the tree. In papaya, the first symptom is a bright yellowing of the upper young leaves; later, symptoms of mosaic, crinkling and leaf tip necrosis develop, followed by a drying of the upper leaves, which progresses to death of the whole plant. Leaf samples showing symptoms from 12 papaya and 6 orange plants were collected from farms in the Melkassa area of east Shewa, during September 2005. Citrus tristeza virus (CTV) has been previously reported in Ethiopia (Khassaye et al., 1977), so citrus samples were tested for CTV by DAS-ELISA, yielding 4/6 positives. DNA was extracted from all plants and indexed in both a nested PCR with phytoplasma generic primers P1/P7 and R16F2n/R16R2, and a simple PCR for papaya bunchy top (PBT) rickettsia using primers PBTF1/PBTR1 (Davis et al., 1998). Nested PCR amplicons were obtained for 11/12 papaya and six citrus samples. Following RFLP analysis, all exhibited identical HaeIII, RsaI and AluI profiles, which were typical of a 16SrII phytoplasma. None of the papaya and citrus samples evaluated was positive for the PBT rickettsia. Sequencing was done by The Sequencing Service (Univ. Dundee, Scotland; http://www.dnaseq.co.uk) using Applied Biosystems Big-Dye Ver 3·1 chemistry, on an Applied Biosystems model 3730 sequencer. The 16S rDNA phytoplasma sequences of a representative sample of papaya (GenBank Acc. No. DQ285659) and citrus (GenBank Acc. No. DQ286576) were identical and their highest similarity (98%) was with papaya yellow crinkle in Australia (GenBank Acc. No. Y10097), a member of the 16SrII group (Candidatus Phytoplasma aurantifolia). This is the first record of phytoplasmas associated with die-back diseases of papaya and citrus in Ethiopia. The finding of a mixed infection of phytoplasma and CTV in some citrus samples will require further studies before any recommendations for disease management can be made. Work in the UK was done under Defra plant health license No. PHF 174D/5185(08/2005). Rothamsted Research receives grant aid from the Biotechnology and Biological Sciences Research Council of the UK.

Detection, Identification and Molecular Characterization of a Phytoplasma Associated with Arabian Jasmine (Jasminum sambac L.) Witches’ Broom in Oman

AbstractArabian jasmine (Jasminum sambac L.) plants showing witches’ broom (WB) symptoms were found in two regions in the Sultanate of Oman. Polymerase chain reaction (PCR) amplification of the 16S rRNA gene and the 16S–23S spacer region utilizing phytoplasma‐specific universal and designed primer pairs, and transmission electron microscopy of phytoplasma‐like structures in phloem elements confirmed phytoplasma infection in the symptomatic plants. PCR products primed with the P1/P7 primer pair were 1804 bp for jasmine witches’ broom (JasWB) and 1805 bp for alfalfa (Medicago sativa L.) witches’ broom (AlfWB). Actual and putative restriction fragment length polymorphic analysis indicated that jasmine and AlfWB phytoplasmas were molecularly indistinguishable from each other and closely related to papaya yellow crinkle (PYC), as well as being distinct from lime WB (LWB) and Omani alfalfa WB (OmAlfWB) phytoplasmas. A sequence homology search of JasWB and AlfWB showed 99.8% similarity with PYC from New Zealand and 99.6% similarity with each other (JasWB/AlfWB). The jasmine and AlfWB phytoplasmas were also shown to be related to the peanut WB group (16SrII) of 16S rRNA groups based on a phylogenetic tree generated from phytoplasma strains primed with the P1/P7 primer pair and representing the 15 phytoplasma groups.

Use of Survival Analysis to Determine the Postincubation Time-to-Death of Papaya Due to Yellow Crinkle Disease in Australia.

The current management recommendation for papaya (Carica papaya) plants exhibiting symptoms of yellow crinkle disease in Australia is the practice of ratooning infected plants. Ratooning involves removing the main stem of diseased papaya plants and allowing a lateral stem (supposedly pathogen-free) to develop and replace the diseased stem. Using nonparametric and parametric methods of survival analysis, we tested different hypotheses regarding plant factors that may influence the postincubation period survival time of phytoplasma-infected papaya. The factors included plant age, the season (wet versus dry) when papaya plants first became symptomatic, and the two predominant phytoplasma strains causing papaya yellow crinkle: tomato big bud (TBB) or sweet potato little leaf strain V4 (SPLL-V4). Median survival time was estimated to be from 4 to 5 months. Therefore, we estimated that the infectious period (incubation period plus the period from postincubation to time-to-death period) of infected papaya ranges from 6 to 9 months. Using parametric accelerated failure modeling and nonparametric Cox proportional hazard modeling, no significant improvement from a null model (no covariates) was found when analyzing plant age, the season a plant was observed to be symptomatic, or phytoplasma strain. However, the season in which a papaya plant became symptomatic differed between the two phytoplasma strains, indicating that the TBB and SPLL-V4 strains may have different modes of insect acquisition and transmission. Because of the long infectious period and the rate of plantto-plant spread, we question the use of ratooning as the primary management tactic for managing papaya yellow crinkle.

Molecular characterization of the 16S rRNA gene of phytoplasmas detected in two leafhopper species associated with alfalfa plants infected with witches' broom in Oman

Two leafhopper species, Austroagallia avicula and Empoasca sp., were consistently found in alfalfa fields infected with witches’ broom phytoplasma (OmanAlfWB) in the Al-Batinah, Dakhliya, North and South Sharqiya, Muscat, and Al-Bureimi regions of the Sultanate of Oman. Phytoplasmas from both leafhoppers were detected by specific polymerase chain reaction (PCR) amplification of the 16S rRNA gene and the spacer region in direct PCR using P1/P7 primer pairs. Comparative RFLP profiles of the amplified rRNA gene and the spacer region from leafhopper phytoplasmas and from 20 phytoplasma controls yielded patterns referable to phytoplasmas belonging to the peanut witches’ broom group (16SrII group). In particular, extensive RFLP analyses with the endonucleases HpaII, Tru9I, Tsp509I, and RsaI indicated that the phytoplasmas in A. avicula and Empoasca sp. were identical but showed some differences from OmanAlfWB; however, RFLP patterns obtained with TaqI showed the OmanAlfWB and the phytoplasmas from the two leafhoppers to be identical. Direct PCR products amplified from phytoplasma leafhopper DNA using the P1/P7 primer pair were cloned and sequenced yielding 1758 bp and 1755 bp products from A. avicula and Empoasca sp. respectively; the homology of these sequences with OmanAlfWB and papaya yellow crinkle phytoplasmas was more than 98%. A phylogenetic tree based on the 16S rRNA gene and spacer region sequences from 44 phytoplasmas revealed that the phytoplasmas from the leafhoppers clustered with OmanAlfWB, papaya yellow crinkle, and gerbera phyllody phytoplasmas, all belonging to 16SrII group, but were distinct from lime witches’ broom phytoplasma, the most commonly found phytoplasma in the Sultanate of Oman.

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.

Epidemiology of Phytoplasma Diseases in Papaya in Northern Australia

Using molecular tools, the spread of phytoplasma diseases in a papaya plantation was investigated for 3 years to identify phytoplasma strains affecting papaya, insect vectors and alternative plant hosts. Five phytoplasma strains (SPLL‐V4, TBB, CaWB, StLL and WaLLvar) were associated with papaya yellow crinkle disease and one phytoplasma strain (PDB) was associated with papaya dieback disease. The most prevalent strains were TBB and SPLL‐V4 which occurred in 94% of infected papaya. There was a significant correlation between phyllody and TBB, and virescence and SPLL‐V4, although other phytoplasma types could also be associated with either phyllody or virescence. No mixed infections were detected in diseased papaya. Disease progress curves for TBB and SPLL‐V4 showed a sigmoid response reaching a maximum disease incidence of 16% after 24 months. The rate of disease spread was best described by a logistic model which showed that TBB spread at a slightly higher rate than SPLL‐V4. Ten phytoplasma strains were detected in 14 alternative plant species; however, TBB and SPLL‐V4 were present in only a few individual plants of some of these species, so these alternative hosts would probably not have provided a significant infection source to papaya. Very few phytoplasmas were detected in leafhoppers collected over 3 years with TBB and SPLL‐V4 only detected in Orosius spp.

Genetic variability in phytoplasmas associated with papaya yellow crinkle and papaya mosaic diseases in Queensland and the Northern Territory

Samples of papaya (Carzca papaya L.) showing symptoms of papaya yellow crinkle (PYC) or papaya mosaic (PM) diseases were collected from two study sites in Queensland (Rockhampton and Caboolture) and from Katherine in the Northern Territory. Phytoplasmas were detected in these samples using polymerase chain reaction (PCR) and fluorescence microscopy techniques. The genetic variability of the phytoplasmas was studied using Southern blot hybridisation, restriction fragment length polymorphism (RFLP), and sequence analysis. RFLP analysis of PYC samples collected from Caboolture indicated that only the tomato big bud (TBB) type phytoplasma was present, whereas both PYC and PM samples collected from Rockhampton exhibited two phytoplasma types, sweet potato little leaf variant (SPLL-V4) and TBB. PYC samples collected from Katherine showed even greater variability with cactus witches’ broom (CWB), which was first described from Indonesia, detected in addition to SPLL-V4 and TBB which are common throughout Australia. Although PYC and PM diseases have different symptoms, no genetically distinct phytoplasma could be found in exclusive association with either disease.

Phylogenetic positions of phytoplasmas associated with dieback, yellow crinkle and mosaic diseases of papaya, and their proposed inclusion in 'Candidatus Phytoplasma australiense' and a new taxon, 'Candidatus Phytoplasma australasia'.

DNA extracted from three papaya (Carica papaya L.) plants, individually affected by dieback, yellow crinkle or mosaic diseases, was subjected to PCR using phytoplasma-specific primers to amplify the 16S rRNA gene plus 16S-23S rRNA intergenic spacer region. Near-complete DNA sequences obtained for the three PCR amplimers were subjected to phylogenetic analyses and direct sequence comparison with other phytoplasma 16S rDNA and 16S-23S spacer region DNA sequences. The papaya yellow crinkle (PpYC) and papaya mosaic (PpM) sequences were identical to each other, but distinctly different from the papaya dieback (PpDB) sequence, showing 90.3% identity in the he 16S rDNA and 87.8% identity in the 16S-23S spacer region DNA sequences. A phylogenetic tree based on 16S rDNA sequences was calculated, in which PpYC and PpM are most closely related to the tomato big bud phytoplasma (TBB; 99.7% 16S rDNA sequence identity) from Australia, within subclade iii. This subclade consists of strains only reported occurring in the Southern Asian region and Australia, which indicates an Asian/Australasian origin. PpDB is most closely related to the Phormium yellow leaf phytoplasma from new Zealand (PYL; 99.9% identity) and the Australian grapevine yellows phytoplasma (AGY; 99.7% identity). These three phytoplasma strains form a distinct clade within subclade xii, which also includes the European strains STOL and VK as another distinct clade. The origin of the closely related but geographically separated AGY-like strains and STOL-like strains of subclade xii is unclear. It is proposed that phytoplasma strains PpDB, PYL and AGY be included in the previously described taxon 'Candidatus Phytoplasma australiense', and that PbYC, PpM and TBB be assigned to a new taxon, "Candidatus Phytoplasma australasia'.

DNA sequence analysis supports the association of phytoplasmas with papaya (Carica papaya) dieback, yellow crinkle and mosaic

Previous studies using the polymerase chain reaction (PCR) with primers that hybridise to regions within and adjacent to the 16S rRNA gene have indicated that Australian papaya dieback, papaya mosaic and papaya yellow crinkle are associated with phytoplasmas (formerly called mycoplasma-like organisms). In this study, DNA sequence analysis of PCR products obtained from affected plant tissue has confirmed that the amplified DNA products are most similar to phytoplasmas associated with other plant diseases. These results support the proposition that phytoplasmas may be the causal agents of these economically significant papaya diseases in Australia. Further, a PCR product obtained from DNA extracts of Rutherglen bugs (Nysius vinitor) found on papaya plants, also had a sequence that was similar to those of other phytoplasmas. However, this sequence was different from any of the PCR products derived from diseased papaya. N. vinitor is an unlikely vector of phytoplasmas because of its mechanism of feeding, but this result suggests that it should be considered in future studies of the vector of papaya dieback, mosaic and yellow crinkle.