Witches' Broom Disease Of Lime Research Articles

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.

Study of erythromycin and copper sulfate effects in controlling witches’ broom disease of lime (WBDL)

Study of erythromycin and copper sulfate effects in controlling witches’ broom disease of lime (WBDL)

Multilocus genotyping of ‘Candidatus Phytoplasma aurantifolia’ associated with witches’ broom disease in Citrus spp.

Witches’ broom disease of lime (WBDL), associated with ‘Candidatus Phytoplasma aurantifolia,’ is a serious disease of acid lime in Southern Iran. In order to characterize ‘Ca. P. aurantifolia’ strains, a multilocus sequence analysis protocol was developed based on different genomic loci, including 16S rRNA, 16S-23S rRNA intergenic spacer (IS), ribosomal protein genes rpl22 and rps3, cell division protein, and group II intron reverse transcriptase/mature gene, in 46 phytoplasma strains. Analyses of virtual RFLP, phylogenetics, and DNA sequence identity of 16S rRNA of Iranian phytoplasma strains associated with symptomatic citrus plants indicated that the Iranian strains were classified in ribosomal subgroup 16SrII-B. In total, three genotypes were recognized based on 16Sr virtual RFLP profiles. Phylogenetic analysis based on the rp and ftsH gene sequences showed clustering of all the strains as one group. Comparative sequence and phylogenetic analysis of group II intron genes indicated the highest differentiation of ‘Ca. P. aurantifolia’–related strains in four genotypes. The polymorphism of group II intron reverse transcriptase/mature genes fragmentation provides finer differentiation of closely related ‘Ca P. aurantifolia’ strains and reveals higher genetic diversity than previously reported.

Changes in nutrient concentrations and biochemical characteristics of Mexican lime (Citrus aurantifolia) infected by phytoplasma

Witches’ broom disease of lime (WBDL), caused by ‘Candidatus Phytoplasma aurantifolia’, is a major limiting factor for Mexican lime (Citrus aurantifolia Swingle) production in southern Iran. Here, nutritional status and biochemical composition of healthy and infected Mexican lime in a greenhouse were compared between uninoculated (healthy) plants and plants 52 weeks after inoculation with the phytoplasma. Leaves from healthy and infected plants were sampled to measure total phenolics, antioxidant capacity, peroxidase and superoxide dismutase enzymatic activities, soluble carbohydrate, and starch. Macro- and microelements were also measured in leaves, stems, and roots of the plants. The results showed that phytoplasma infection led to a marked increase in soluble carbohydrates and starch in the leaves, but to a reduction in the roots. In leaves and roots, antioxidant capacity, phenolics, and peroxidase activity were enhanced after infection; nitrogen, calcium, boron, and manganese were significantly lower and phosphorus, magnesium, potassium, iron, zinc, and copper significantly higher in the infected leaves. This study provides new insights on metabolic and nutritional changes associated with WBDL in the greenhouse.

“Witches’ broom” disease of lime suppressed by some resistance inducers

“Witches’ broom” disease of lime (WBDL) has been considered as one of the most economically devastating diseases of Mexican lime in southern Iran with poor information on the management and almost no available cure. Resistance inducers are known as chemical compounds that enable plant defense system to suppress pathogens. In this study the effects of ascorbic acid (300 µM); ascorbic acid (600 µM), ascorbic acid (300 µM) plus MgCl2 (200 µM), salicylic acid (150 µM); salicylic acid (300 µM), salicylic acid (300 µM) plus MgCl2 (200 µM), Phosetyl aluminum (Aliette) (2/000), Nano silver (10 µM) and STARNER™ (3 g/L), 31% Phosetyl-Al plus 53% propamocarb (Privicure Energy™) (17.5 µM), Hymexazol 70% (Tachigaren™) (30 µM) and NORDOX(30 µM) spray on 2-years-old seedlings infected WBDL were investigated at greenhouse and field conditions simultaneously. Disease severity data were recorded during seven visual evaluations for greenhouse experiment and five evaluations time points for the field experiment. To determine disease severity, each tree was divided into three sections, and each zone was scored individually based on the visual symptoms from 0 to 5. Also, the fruit yield data were collected for the two harvest seasons in Minab, Hormozgan province of Iran. A significant suppression effect on WBDL was observed after spraying 31% of Phosetyl aluminum plus 53% propamocarb (Privicure Energy™) and 70% of Hymexazol separately two times per year. Ascorbic acid, salicylic acid, Aliette, NanoSilver and STARNER also showed transient effect against WBDL after three or four applications per year. The treatments of 31% Phosetyl-Al plus 53% propamocarb (Privicure Energy™) 4/000, Hymexazol 70% (2/000) and NORDOX (0.5/000) decreased WBDL severity by approximately 10 to 40% in the greenhouse and 25% in the field. The average number of fruit per tree was 2300.1 fruit/tree, while that of the negative control was only 1800.5 fruit/tree. Due to positive effect of these resistance inducer compounds on WBDL severity, fruit yield significantly increased in infected lime orchards. The results of this study indicate some promising signs of induced resistance for suppressing the severity of the disease in WBDL infected orchards.

Reaction of some commercial citrus species and Iranian lime biotypes to witches' broom disease of lime

Witches' broom disease of lime (WBDL), associated with Candidatus Phytoplasma aurantifolia, has devastated the majority of Mexican lime orchards in southern Iran. Recognition of resistant cultivars and rootstocks can play a major role in WBDL management. In this study, graft-inoculation was used to address the reaction of 12 citrus commercial species and 39 collected lime biotypes from five main lime production areas of Hormozgan province, Iran, against WBDL phytoplasma. The experimental plants were allowed to grow from seed, then five seedlings of each species and biotypes were grafted with Ca. Phytoplasma aurantifolia infected shoots. Inoculated plants were inspected for symptom expression for two years. Direct and nested-PCR using P1/P7 and R16F2n/R16R2 primer pairs, respectively, were used for confirmation of phytoplasmal infection in the suspected plants. All commercial citrus species and 39 of the lime biotypes showed the symptoms of little leaf, internode shortening and witches' broom after two to twenty months from graft-inoculation. Incubation period in plants varied from two months in Astaraei lemon and R11 biotype of Rudan region to twenty months in S4 and S6 biotypes of Senderk region. With these results, we can say all commercial citrus species were sensitive to WBDL. Among the commercial citrus species, Lisbon lemon, grapefruit and Cleopatra mandarin cultivars showed symptoms of WBDL after 460, 425 and 375 days, respectively. Disease symptoms developed rapidly on Astaraei lemon and Bakraei within 85–105 days, respectively. Among the collected biotypes, one, five and one biotypes from Minab, Rudan and Hashtbandi, respectively, were resistant to WBDL phytoplasma. These biotypes were symptomless; however, the PCR assay for detection of the phytoplasma was positive in all of them. These results are promising regarding the potential of using the suitable biotypes to reduce the development of WBDL.

Genetic Variation of Lime (Citrus sp.) Accessions Using Flow Cytometry Technique, Morphological Characteristics and Molecular Markers

Because of sexual propagation of Mexican lime in southern regions of Iran, there are many lime accessions (lime biotypes) in Mexican lime gardens in which appear some variation in fruit and tree shape. However, most of these accessions are susceptible to witches broom disease of lime (WBDL). Persian lime (C. latifolia Tanaka) is a triploid WBDL-tolerant species. Considerable number of un-described lime biotypes have been cultivated by farmers as Persian lime with no documented evidence related to their origin and genetic background. To unveil systematic modality of the lime accessions, we investigated 18 lime accessions collected from Fars, Hormozgan and Mazandaran provinces (Iran) using flow cytometry (FCM) approach, morphological characteristics, ISSR and SSR markers in 2016. The results of FCM revealed that 14 out of 18 lime accessions were triploid. Triploid accessions showed higher leaf dimension values together with lower stomata and secretory sacs density compared with diploid accessions. Application of SSR markers confirmed the obtained results of ploidy level determination using FCM and morphological analysis. Results of morphological and ISSR markers demonstrated that four out of 18 accessions were different from others and it was in correspondence with the results of FCM. SSR results also grouped the accessions in line of FCM. In conclusion, it has been shown that IFJKh, Cucumber-shaped lime and IFJKMes accessions, are diploid and their cultivation is not recommended in the infected regions to WBDL phytoplasma.

Differential expression and phytohormone unbalance in Citrus aurantifolia plants during “sudden decline of lime”, a new phytoplasma disease of citrus

For decades, the production of acid lime (Citrus aurantifolia) in Oman has been affected by diseases caused by phytoplasmas, notably witches’ broom disease of lime (WBDL). In recent years, a new phytoplasma strain in Oman has been observed that promotes the sudden decline of lime (SDL). The molecular mechanisms behind its pathogenicity and mode of interaction with citrus host plants is still completely unknown. In this study, we evaluated the differential expression of genes in symptomatic and asymptomatic lime trees in Oman using a real-time quantitative PCR assay. Among 27 regulatory and biosynthesis-related genes tested in Citrus aurantifolia plants during phytoplasma infection, we verified the presence of 14 responsive genes in plants showing SDL symptoms, revealing a specific set of SDL-responsive genes. Quantification data of endogenous 3-indoleacetic acid and jasmonic acid show an unbalanced hormonal content in symptomatic lime trees, corroborating the gene expression data. The identification of regulatory genes differentially expressed in plant-phytoplasma interactions during SDL will help to elucidate the mechanisms possibly involved in defense responses, development and death-triggered signals in infected citrus plants.

Identification of putative effector genes and their transcripts in three strains related to ‘Candidatus Phytoplasma aurantifolia’

Molecular mechanisms underlying phytoplasma interactions with host plants are largely unknown. In this study attempts were made to identify effectors of three phytoplasma strains related to ‘Ca. P. aurantifolia’, crotalaria phyllody (CrP), faba bean phyllody (FBP), and witches’ broom disease of lime (WBDL), using information from draft genome of peanut witches’ broom phytoplasma. Seven putative effectors were identified in WBDL genome (SAP11, SAP21, Eff64, Eff115, Eff197, Eff211 and EffSAP67), five (SAP11, SAP21, Eff64, Eff99 and Eff197) in CrP and two (SAP11, Eff64) in FBP. No homologs to Eff64, Eff197 and Eff211 in phytoplasmas of other phylogenetic groups were found. SAP11 and Eff64 homologs of ‘Ca. P. aurantifolia’ strains shared at least 95.9% identity and were detected in the three phytoplasmas, supporting their role within the group. Five of the putative effectors (SAP11, SAP21, Eff64, Eff115, and Eff99) were transcribed from total RNA extracts of periwinkle plants infected with these phytoplasmas. Transcription profiles of selected putative effectors of CrP, FBP and WBDL indicated that SAP11 transcripts were the most abundant in the three phytoplasmas. SAP21 transcript levels were comparable to those of SAP11 for CrP and not measurable for the other phytoplasmas. Eff64 had the lowest transcription level irrespective of sampling date and phytoplasma isolate. Eff115 transcript levels were the highest in WBDL infected plants. This work reports the first sequence information for 14 putative effectors in three strains related to ‘Ca. P. aurantifolia’, and offers novel insight into the transcription profile of five of them during infection of periwinkle.

First report of a 'Candidatus Phytoplasma aurantifolia'‐related strain associated with witches'‐broom disease of limequat in Iran

Witches'-broom disease of lime (WBDL) caused by 'Candidatus Phytoplasma aurantifolia' was found first in Mexican lime ( Citrus aurantifolia ) in a small area in southern Iran in 1997 (Bove et al ., 2000). Since then WBDL has spread to …

Invasive mutualisms between a plant pathogen and insect vectors in the Middle East and Brazil.

Complex multi-trophic interactions in vectorborne diseases limit our understanding and ability to predict outbreaks. Arthropod-vectored pathogens are especially problematic, with the potential for novel interspecific interactions during invasions. Variations and novelties in plant–arthropod–pathogen triumvirates present significant threats to global food security. We examined aspects of a phytoplasma pathogen of citrus across two continents. ‘Candidatus Phytoplasma aurantifolia’ causes Witches' Broom Disease of Lime (WBDL) and has devastated citrus production in the Middle East. A variant of this phytoplasma currently displays asymptomatic or ‘silent’ infections in Brazil. We first studied vector capacity and fitness impacts of the pathogen on its vectors. The potential for co-occurring weed species to act as pathogen reservoirs was analysed and key transmission periods in the year were also studied. We demonstrate that two invasive hemipteran insects—Diaphorina citri and Hishimonus phycitis—can vector the phytoplasma. Feeding on phytoplasma-infected hosts greatly increased reproduction of its invasive vector D. citri both in Oman and Brazil; suggesting that increased fitness of invasive insect vectors thereby further increases the pathogen's capacity to spread. Based on our findings, this is a robust system for studying the effects of invasions on vectorborne diseases and highlights concerns about its spread to warmer, drier regions of Brazil.

Population genetic analysis reveals a low level of genetic diversity of 'Candidatus Phytoplasma aurantifolia' causing witches' broom disease in lime.

Witches’ broom disease of lime (WBDL) is a serious phytoplasma disease of acid lime in Oman, the UAE and Iran. Despite efforts to study it, no systemic study attempted to characterize the relationship among the associated phytoplasma, ‘Candidatus Phytoplasma aurantifolia’, from the three countries. This study utilized sequences of the 16S rRNA, imp and secA genes to characterize 57 strains collected from Oman (38), the UAE (9) and Iran (10). Phylogenetic analysis based on the 16S rRNA gene showed that the 57 strains shared 98.5–100 % nucleotide similarity to each other and to strains of ‘Ca. P. aurantifolia’ available in GenBank. The level of genetic diversity was low based on the 16S rRNA (0-0.011), imp (0–0.002) and secA genes (0–0.015). The presence of low level of diversity among phytoplasma strains from Oman, the UAE and Iran can be explained by the movement of infected lime seedlings from one country to another through trading and exchange of infected plants. The study discusses implication of the findings on WBDL spread and management.

Increased sodium and fluctuations in minerals in acid limes expressing witches' broom symptoms.

Witches’ broom disease of lime (WBDL), caused by ‘Candidatus Phytoplasma aurantifolia’, is a very serious disease of acid limes. The disease destroyed more than one million lime trees in the Middle East. WBDL results in the production of small, clustered leaves in some branches of lime trees. Branches develop symptoms with time and become unproductive, until the whole tree collapses within 4–8 years of first symptom appearance. This study was conducted to investigate differences in minerals between symptomatic and asymptomatic leaves of infected lime trees. The study included one set of leaves from uninfected trees and two sets of infected leaves: symptomatic leaves and asymptomatic leaves obtained from randomly selected acid lime trees. Nested polymerase chain reaction detected phytoplasma in the symptomatic and asymptomatic leaves from the six infected trees, but not from the uninfected trees. Phylogenetic analysis showed that all phytoplasmas belong to the 16S rRNA group II-B. Mineral analysis revealed that the level of Na significantly increased by four times in the symptomatic leaves compared to the non-symptomatic leaves and to the uninfected leaves. In addition, symptom development resulted in a significant increase in the levels of P and K by 1.6 and 1.5 times, respectively, and a significant decrease in the levels of Ca and B by 1.2 and 1.8 times, respectively. There was no significant effect of WBDL on the levels of N, Cu, Zn, and Fe. The development of witches’ broom disease symptoms was found to be associated with changes in some minerals. The study discusses factors and consequences of changes in the mineral content of acid limes infected by phytoplasma.

Chemical composition of acid lime leaves infected with <i>Candidatus</i> Phytoplasma aurantifolia

The production of acid lime (Citrus aurantifolia) has declined in many parts of the world due to phytoplasmal infection by “Candidatus Phytoplasma aurantifolia”. The resulting Witches’ Broom Disease of Lime (WBDL) causes stem and leaf proliferation and clustering that starts on a few branches and continues to spread until trees are killed within 5-7 years. Recent studies have shown that Phytoplasma alters the chemical composition of leaves. Leaves from WBDL-symptomatic lime trees were collected to determine their volatile compound composition. Phytoplasmal infection was confirmed by a polymerase chain reaction (PCR) assay using primers P1/P7 and R16F2n/R16R2 in direct and nested PCR, respectively. Restriction fragment length polymorphism (RFLP) profiles of acid lime Phytoplasma were identical with those of WBDL Phytoplasma. The phytochemical composition of symptomatic (infected) and asymptomatic (healthy) leaves of acid lime were determined using GC-MS analysis of steam distilled extract. The WBDL-symptomatic leaves had higher concentration in ?-limonene, β-ocimene and trans-caryophyllene and a reduction in other compounds (i.e. citral, citronellal, cisverbenol, neryl acetate, and linalool). Variations in the leaf phytochemical concentration indicate a possible role in the development of the WBDL disease symptoms.

Detection of Candidatus Phytoplasma aurantifolia with a quantum dots fret-based biosensor.

The witches’ broom disease of lime (WBDL) supposedly caused by Candidatus Phytoplasma aurantifolia (Ca. P. aurantifolia) is the most devastating disease of lime in Oman and southern parts of Iran. The present study describes developing a quantum dot (QD)-based nano-biosensor for the highly sensitive detection of phytoplasma in infected trees. The immunodominant membrane protein (IMP) expressed in the surface of phytoplasma was selected as a target protein for construction of a specific binding antibody. The antibody (anti-IMP) was effectively conjugated to tioglicolic acid-modified cadmium-telluride quantum dots (CdTe-QDs) synthesized in an aqueous solution via electrostatic interaction. Dye (rhodamine) molecules were attached to the IMP, then, the donor-acceptor complexes (QDs-Ab-IMPRhodamine) were formed based on the antigen-antibody interaction. The mutual affinity of the antigen and the antibody brought the CdTe QDs and rhodamine together close enough to allow the fluorescence resonance energy transfer (FRET) to occur. The immunosensor constructed showed a high sensitivity and specificity of 100%, a detection limit of 5 ca. P. aurantifolia/µl, and acceptable stability, so it could be used for detection with consistent results.

AFLP data suggest a potential role for the low genetic diversity of acid lime (Citrus aurantifolia Swingle) in Oman in the outbreak of witches’ broom disease of lime

Acid lime (Citrus aurantifolia) is the fourth largest fruit crop in terms of cultivated area and production in Oman. However, over half a million lime trees were lost in Oman over the past 35 years due to witches’ broom disease of lime (WBDL) which is caused by Candidatus phytoplasma aurantifolia. This study was conducted to examine genetic diversity of acid lime in Oman. AFLP analysis of 143 acid lime samples from Oman, 2 from Brazil and one from Pakistan using 4 primer pair combinations produced 980 polymorphic loci (100 %) and 146 AFLP genotypes. Despite the long history of acid lime cultivation in Oman, populations of lime from different districts were found to have low levels of genetic diversity (0.0888–0.2284). AMOVA analysis indicated the existence of high level of genetic differentiation (FST = 0.271) among populations of acid lime from Oman and Brazil, which indicates that both populations have evolved independently for a considerably long period of time. On the other hand, AMOVA analysis showed that only 11 % of the genetic variation exists among populations from the 18 different districts in Oman. This suggests frequent exchange of acid lime planting material across geographical regions in Oman. Findings from this study suggest that the low level of genetic diversity of acid lime in Oman and frequent movement of acid lime planting material across districts are two main factors which contributed to the rapid spread and high susceptibility of acid limes to WBDL in the country.

Witches'-Broom Disease of Lime Affects Seed Germination and Seedling Growth But Is Not Seed Transmissible

Witches'-broom disease of lime (WBDL), caused by 'Candidatus Phytoplasma aurantifolia', has devastated many Mexican lime orchards and is currently a threat to lime production in neighboring provinces of southern Iran. Several reports have suggested transmission and spread of WBDL phytoplasma through the seed of infected plants. In the present study, claims of seed transmission of this phytoplasma were examined. Fruit were collected from infected trees in the infested areas of Minab (Hormozgan Province) and from symptomless trees in noninfested areas. Lime seed from symptomless and witches'-broom-affected trees were sown in separate beds in an insect-proof screenhouse and the resulting seedlings were examined for phytoplasmal infection. Leaf, stem, and root samples were collected from both groups of seedlings every 3 months for 2 years and tested for WBDL phytoplasma using direct and nested polymerase chain reaction (PCR). Repeated PCR tests on the seedlings did not reveal the presence of phytoplasmal DNA. Likewise, symptoms of the disease were not observed on these seedlings after 2 years. PCR assays detected the phytoplasma in coats of some seed from infected trees; however, no excised embryos were positive for the phytoplasma. All positive PCR results were confirmed by restriction fragment length polymorphism assay. One-year-old seedlings derived from seed of noninfected plants appeared more vigorous in terms of height, number of leaves, and fresh weight of shoot compared with those from infected trees. The germination percentage, mean daily germination, peak value, and germination value were significantly higher for seed of fruit from noninfected trees and seed from fruit on asymptomatic branches of infected trees than those from fruit on symptomatic branches of infected trees.

Control of Huanglongbing (HLB) disease with reference to its occurrence in Malaysia

The center of diversity for citrus was originally found on the northeastern India, eastward through the Malay Archipelago and south to Australia. Today citrus is produced in 140 countries, mainly between the north and south 40o latitudes. Citrus ranked first among fruit crops in the international trade based on value. Citrus production of the world is around 105 million tons per year. Orange ( Citrus sinensis ) accounts for almost two thirds of the total citrus production (65%), followed by tangerine ( Citrus reticulata ) (21%), lemon ( C. limon ) (6%) and grapefruit ( C. paradisi ) (5.5%). Other significant commercially grown species are lime ( C. aurantifolia ), pummelo ( C. grandis ) and citron ( C. medica ). The largest citrus producers are Brazil (20%), United States (14%), China (12%), Mexico (6%) and the countries of the mediterranean basin (15%). Humidity and day-to-night temperature fluctuations influence which varieties are best adapted to an area. Most citrus fruits are produced for fresh market consumption and only around 30% is processed. Fresh fruits are rich in vitamin C which plays a vital role in prevention of scurvy. After extraction of the juice, the skin and fruit pulp can be used as livestock feed or making compost. The rind acid (oil) of the citrus is considered an expensive commodity in international trade (F.A.O 2003). Unfortunately, the citrus industry is threatened by 2 destructive diseases namely Witches’ Broom disease of lime (WBDL) and Huanglongbing (HLB) disease. WBDL has been known to be caused by Candidatus phytoplasma aurantifolia . It is one of the destructive diseases on citrus industry in the Middle East, India and Pakistan. It was reported in 1970 for the first time from lime orchard of Oman, united Arabian emirate (UAE) (1988), Iran (1998) and India and Pakistan (1999). WBDL is a phloem limited phytoplasma disease of lime. HLB disease causing citrus greening ( Candidatus liberibacter spp.) is the second most severe disease on citrus industry all over the world. HLB has destroyed an estimated 60 million trees in Africa and Asia. More than 40 countries were infected by HLB in Africa, Asia and USA (Chau et al., 1996; Bove, 2006; Roux et al., 2006; Batool et al., 2007). The HLB pathogens are highly fastidious phloem-inhabiting bacteria in the genus Candidatus liberibacter . The bacteria have not been cultured yet in laboratory media and do not survive outside the host cells. Three types of phloem limited bacteria causing HLB disease have been described and identified (Bove, 2006). The isolate from South Africa ( Candidatus liberibacter africanus ) is considered heat-sensitive and found in Africa. It is vectored by African citrus psyllid ( Trioza erytreae) and was described by Guercio in 1918. The isolate from Asia ( Candidatus liberibacter asiaticus ) is more severe and widespread; it is vectored by Asian psyllid (Diaphorina citri Kuwayama) (Garnier et al., 2000). This type of HLB is heat-tolerant (Garnier et al., 2000). It can show the symptom on humid, cool and hot temperature, up to 35oC (Garnier et al., 2000; Bove 2006; Le Roux et al., 2006). The isolate from America has been named Candidatus liberibacter americanus ; it was detected in Brazil and Florida (Coletta-Filho et al., 2005; Texeira et al., 2005).

First report of citrus huanglongbing disease on orange in Iran

Plant PathologyVolume 58, Issue 4 p. 793-793 Free Access First report of citrus huanglongbing disease on orange in Iran M. M. Faghihi, M. M. Faghihi Hormozgan Agriculture and Natural Resources Research Centre;Search for more papers by this authorM. Salehi, M. Salehi Fars Agriculture and Natural Resources Research Centre; andSearch for more papers by this authorA. Bagheri, A. Bagheri Hormozgan Agriculture and Natural Resources Research Centre;Search for more papers by this authorK. Izadpanah, Corresponding Author K. Izadpanah Department of Plant Protection, College of Agriculture, Shiraz University, Shiraz, Iran *E-mail: izadpana@shirazu.ac.ir. Accepted 23 December 2008 at http://www.bspp.org.uk/ndr where figures relating to this paper can be viewed.Search for more papers by this author M. M. Faghihi, M. M. Faghihi Hormozgan Agriculture and Natural Resources Research Centre;Search for more papers by this authorM. Salehi, M. Salehi Fars Agriculture and Natural Resources Research Centre; andSearch for more papers by this authorA. Bagheri, A. Bagheri Hormozgan Agriculture and Natural Resources Research Centre;Search for more papers by this authorK. Izadpanah, Corresponding Author K. Izadpanah Department of Plant Protection, College of Agriculture, Shiraz University, Shiraz, Iran *E-mail: izadpana@shirazu.ac.ir. Accepted 23 December 2008 at http://www.bspp.org.uk/ndr where figures relating to this paper can be viewed.Search for more papers by this author First published: 20 July 2009 https://doi.org/10.1111/j.1365-3059.2009.02051.xCitations: 16AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Huanglongbing (HLB), also known as greening, is a destructive disease of citrus that may limit production of this crop in many parts of Asia, Africa and South America. HLB is caused by a non-culturable phloem-limited bacterium of the genus ‘Candidatus Liberibacter’. Three Candidatus species of Liberibacter, namely, ‘Ca. L. asiaticus’, ‘Ca. L. africanus’ and ‘Ca. L. americanus’, have been identified (Bové, 2006). The psyllid Diaphorina citri, vector of ‘Ca. L. asiaticus’ was first detected in December 1997 in Iran in an area close to the border with Pakistan (Bovéet al., 2000). Since then, high populations of D. citri have been found in citrus plantations of Hormozgan and Kerman provinces in southern Iran (Bové, 2006). Citrus trees in this region exhibit typical HLB symptoms including mottling of leaves and yellowing of shoots. In the present study leaf samples from Valencia sweet orange trees (Citrus sinensis) (20 with and 20 without symptoms) and over 50 psyllid samples were collected from various locations in Sistan-Baluchistan and Hormozgan provinces and total DNA was extracted using the CTAB (cetyltrimethylammonium bromide) method. DNA samples were tested for presence of ‘Ca. L. asiaticus’ by nested PCR using primer pairs F1 (5′-TGAATTCTTCGAGGTTGGTGAGC-3′)/R1(5′-AGAATTCGACTTAATCCCCACCT-3′) as the first set and F2(5′-GCGTTCATGTAGAAGTTGTG-3′)/R2(5′-CCTACAGGTGGCTGACTCAT-3′) as the second set. Both primer pairs were designed based on the published beta-operon of ribosomal protein encoding sequence of ‘Ca. L. asiaticus’ (Villechanoux et al., 1993). Nested PCR resulted in amplification of a 400 bp product from five out of 20 leaf samples and 26 out of 50 psyllid samples. No amplicons were obtained from symptomless sweet orange trees and psyllid samples reared on healthy sweet orange seedlings. The amplified fragment from a psyllid sample was cloned and sequenced (GenBank Accession No. FJ172759). A blast search showed 100% identity with corresponding sequences of ‘Ca. L. asiaticus’ (M9439, AY34200 and EU078703). This is the first report on the occurrence of HLB disease in Iran. The disease appears to be widely distributed in citrus growing regions of southern Iran and may be a component of citrus decline in this area. References Bové JM, 2006. Huanglongbing: a destructive, newly–emerging, century-old disease of citrus. Journal of Plant Pathology 88, 7– 37. Web of Science®Google Scholar Bové JM, Danet GL, Bananej K et al ., 2000. Witches’ broom disease of lime (WBDL) in Iran. In: RH Yokomi, JV Da Graça, RF Lee, eds. Proceedings of the 14th Conference of the International Organization of Citrus Virologists. Riverside, CA, USA: IOCV, 300– 9. Google Scholar Villechanoux S, Garnier M, Laigret F, Renaudin J, Bové JM, 1993. The genome of the non-cultured, bacterial-like organism associated with citrus greening disease contains the nusG-rplKAJL gene cluster and the gene for a bacteriophage type DNA polymerase. Current Microbiology 26, 161– 6. CrossrefCASPubMedWeb of Science®Google Scholar Citing Literature Volume58, Issue4August 2009Pages 793-793 ReferencesRelatedInformation

Transmission of 'Candidatus Phytoplasma aurantifolia' to Bakraee (Citrus reticulata Hybrid) by Feral Hishimonus phycitis Leafhoppers in Iran.

Witches'-broom disease of lime (WBDL) caused by 'Candidatus Phytoplasma aurantifolia' is a devastating disease in the Sultanate of Oman, United Arab Emirates, and southern Iran. The disease primarily affects lime (Citrus aurantifolia), but in Iran, it is also found in bakraee, a natural C. reticulata hybrid. The disease has been experimentally transmitted from lime to several citrus cultivars by grafting and to a number of herbaceous hosts by dodder. However, the natural vector of 'Ca. P. aurantifolia' has not been determined. The most common phloem-feeding insect associated with lime trees in the area is the leafhopper Hishimonus phycitis. The WBDL phytoplasma has been detected in the body of this leafhopper by ELISA and PCR (1), but previous attempts to establish its vector status have failed. It was recently reported that the leafhopper can release the phytoplasma into a sugar solution by feeding through a Parafilm membrane (4). Here we report successful transmission of WBDL phytoplasma to bakraee seedlings by H. phycitis. The leafhopper nymphs and adults were collected in a WBDL-infected lime orchard in Minab (Hormozgan Province) in May of 2006. Of more than 100 leafhopper samples tested, at least 70% were positive for the phytoplasma by PCR using P1/P7 primer pair (3). Additional field-collected leafhoppers were caged (five per plant) on bakraee seedlings at the two-leaf stage in pots in the greenhouse in Zarghan (Fars Province). After 8 weeks, the remaining leafhoppers were killed with an insecticide. Six months after inoculation, 3 of 10 inoculated plants showed typical symptoms of WBDL, including bud proliferation, general chlorosis, and stunting. Symptomatic plants were strongly positive in PCR assays using primer pair P1/P7. No amplification was obtained with healthy control lime or nonsymptomatic bakraee seedlings. Amplified P1/P7 primed PCR products (1,800 bp) from experimentally vector-challenged bakraee seedlings, captured H. phycitis, and a naturally infected lime tree from Minab were subjected to restriction fragment length polymorphism (RFLP) analysis using AluI, HhaI, HpaII, RsaI, and TaqI enzymes. RFLP patterns from these sources were identical and similar to those reported earlier (2). These analyses verified the identity of WBDL phytoplasma in experimentally infected bakraee seedlings. To our knowledge, this is the first report of natural transmission of 'Ca. P. aurantifolia' by H. phycitis.