Broom Disease Of Lime Research Articles

Optimal management of citrus disease and extreme climatic factors

This study develops a dynamic bioeconomic model using the Bellman equation to derive the optimal management strategy for Witches’ Broom Disease of Lime (WBDL) and the optimal replanting age of citrus trees after the first attack under multiple scenarios of disease management options and climate conditions. In addition, the study investigates different scenarios of disease presence and spread given different climatic conditions of arid, semi-arid and tropical zones in Oman. Factors such as disease incidence, severity, spread, and specific climatic conditions of three growing regions are considered. The farmers' optimal disease mitigation decision is determined by the disease density and insect vector level on the farm, and adverse climate conditions have a significant negative impact on the economic value of citrus cultivation. Results indicate that management measures by citrus growers and policymakers should be based on the climatic conditions of the growing region with a high level of treatment frequency and intensity in the arid region, and lime production should be concentrated in the tropical and semi-arid regions of the country.

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.

Optimal management of witches’ broom disease of lime in presence of extreme environmental factors using stochastic dynamic modelling

Optimal management of witches’ broom disease of lime in presence of extreme environmental factors using stochastic dynamic modelling

Reaction of some Persian lime accessions on different rootstocks to witches’ broom disease of lime

Reaction of some Persian lime accessions on different rootstocks to witches’ broom disease of lime

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.

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.

Witch's Broom Disease of Lime (Candidatus Phytoplasma aurantifolia): Identifying High-Risk Areas by Climatic Mapping.

Biological invasions of vectorborne diseases can be devastating. Bioclimatic modeling provides an opportunity to assess and predict areas at risk from complex multitrophic interactions of pathogens, highlighting areas in need of increased monitoring effort. Here, we model the distribution of an economically critical vectorborne plant pathogen 'Candidatus Phytoplasma aurantifolia', the etiological agent of Witches' Broom Disease of Lime. This disease is a significant limiting factor on acid lime production (Citrus aurantifolia, Swingle) in the Middle East and threatens its production globally. We found that temperature, humidity, and the vector populations significantly determine disease distribution. Following this, we used bioclimatic modeling to predict potential novel sites of infections. The model outputs identified potential novel sites of infection in the citrus producing regions of Brazil and China. We also used our model to explore sites in Oman where the pathogen may not be infectious, and suggest nurseries be established there. Recent major turbulence in the citrus agricultural economy has highlighted the importance of this work and the need for appropriate and targeted monitoring programs to safeguard lime production.

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.

Genetic analysis of 'Candidatus Phytoplasma aurantifolia' associated with witches' broom on acid lime trees.

“Candidatus Phytoplasma aurantifolia” is associated with witches’ broom disease of lime in Oman and the UAE. A previous study showed that an infection by phytoplasma may not necessarily result in the physical appearance of witches’ broom symptoms in some locations in Oman and the UAE. This study investigated whether phytoplasma strains belonging to “Ca. P. aurantifolia” (based on the 16S rRNA gene analysis) in locations where disease symptoms are expressed are different from phytoplasma in locations where disease symptoms are not expressed. About 21 phytoplasma strains (15 from areas and trees with disease symptoms and six from areas and trees without disease symptoms) were included in the analysis. The study utilized sequences of the imp and SAP11 genes to characterize the 21 strains. Phylogenetic analysis of both genes showed that the 21 strains are similar to each other and to reference strains in GenBank. The study shows that there is a low level of diversity among all phytoplasma strains. In addition, it shows that phytoplasma in places where witches’ broom symptoms are not expressed are similar to phytoplasma in places where disease symptoms are expressed. This may suggest that disease expression is not linked to the presence of different phytoplasma strains, but may be due to other factors such as weather conditions.

Pest categorisation of Witches' broom disease of lime (Citrus aurantifolia) phytoplasma.

The EFSA Panel on Plant Health performed a pest categorisation for the Witches' broom disease of lime (Citrus aurantifolia) phytoplasma for the EU territory. The pest has been reported in a few countries in the Middle East and is not known to occur in the EU. The disease is caused by a well‐defined phytoplasma strain in the ‘Candidatus Phytoplasma aurantifolia' species, for which efficient molecular detection assays are available. The most important known natural host is Citrus aurantifolia, which is only grown for ornamental purposes in the EU. Sweet limes, rough lemon and trifoliate orange are also naturally infected by that phytoplasma. The latter can be transmitted by grafting also to some citrus species. Other citrus species were reported to be resistant; however, their susceptibility has been assessed only by symptom observations, and the possible presence of phytoplasmas in symptomless plants cannot be ruled out. The phytoplasma is transmitted by the leafhopper Hishimonus phycitis, which is not known to occur in the EU. There is no information on the vector status of other phloem feeding insects of citrus present in the EU. The pest is listed in Annex IIAI of Directive 2000/29/EC. The main pathways for entry, plants for planting and the vector insect, are closed by existing legislation on import of citrus plants. Nevertheless, should the pest enter, it could establish and spread. In countries where Witches' broom disease of lime (WBDL) is present, it has significant impact. The main knowledge gaps concern (1) and vertical transmission of the phytoplasma to H. phycitis eggs (2) lack of information regarding susceptibility of citrus crops grown in the EU (3) status of potential insect vectors in the EU. Therefore, the WBDL phytoplasma meets the criteria assessed by EFSA for consideration as a potential Union quarantine pest.

Joseph (Josy) M. Bové Dedication

5th International Research Conference on Huanglongbing, Florida, 2017 Joseph (Josy) M. Bove dedication J Ayres 1 , LW Timmer 2 , and T Gottwald 3 Fundecitrus, SP, Brazil; 2 University of Florida, FL, USA; 3 USDA, FL, USA. Citation: Ayres J, Timmer LW, Gottwald T. 2017. Joseph (Josy) M. Bove dedication. J Cit Pathol. iocv_journalcitruspathology_34815. Joseph (Josy) M. Bove was a consummate professional who made many important discoveries that have had a significant effect on plant pathology and on citrus industries worldwide. He was born in Luxemburg, in 1929, moved to France as a boy and became a French citizen in 1968. He did his doctoral work on the in vitro synthesis of plant viral RNA under Jacques Monod at the Pasteur Institute, but most of his career was dedicated to the investigation of systemic prokaryotes infecting citrus. He was Researcher at the French Institute for Citrus and Tropical Fruit Research at Versailles, France (1959-1970); iocv_journalcitruspathology_34815 Director of Research at INRA (French National Institute for Agricultural Research) campus of Bordeaux, France (1971-1975) and professor of microbiology at the University of Bordeaux (1976-1997), Head of Laboratory for Cellular and Molecular Plant Biology (1974-1994) and President of INRA-Bordeaux (1984-1994). Two of his most important discoveries were that the agent of citrus stubborn disease was not a virus as thought previously, but a bacterium and, more precisely, a helical mycoplasma to be named Spiroplasma citri. Similarly, the agent of citrus greening disease was not a virus, but also a bacterium, to be named Candidatus Liberibacter africanus for the disease in Africa and Ca. L. asiaticus for the disease in Asia. His group used antibiotic therapy and electron microscopy to demonstrate that the pathogen was a gram-negative bacterium. He also found that witches’ broom disease of lime, a disease that was devastating lime production in the Middle East, was associated with a mycoplasma Ca. Phytoplasma aurantifolia and transmitted by a leafhopper. Many colleagues and students were initially intimidated by his strong personality and his self- confidence, and he was often critical of weak papers and statements not supported with good data. But, he was always helpful to all, provided valuable suggestions, and was willing to share his knowledge and undertake projects of international cooperation. One of his major and unusual strengths was his ability to link the latest scientific findings with solutions to agricultural problems in the field. During his academic career, Bove published over 300 scientific articles in high impact journals. He served as Chairman of the International Organization of Citrus Virology and the International Organization of Mycoplasmology. He organized many scientific meetings and hosted numerous scientists and trainees in his lab and at his home with his wife Collette, who was a great scientist in her own right. Josy was a consultant for FAO on citrus diseases and traveled extensively. He was widely known everywhere and greatly respected for his insight into all types of problems. His list of countries visited was much longer than the ones he missed.

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.

A new disease inCitrus aurantifoliain Oman, “sudden decline”, is associated with a pathogen complex including a 16SrII group phytoplasma

Witches’ broom disease of lime is a devastating disease in the Sultanate of Oman. In recent years a new disease has been observed, which promotes a sudden decline in Citrus aurantifolia (acid lime). The etiological agent of this new disease has not yet been determined. In this study the presence of phytoplasmas and RNAs was evaluated in plants showing the sudden decline symptomatology. The results reveal the presence of a complex of at least three different pathogens: 16SrII phytoplasmas, Citrus tristeza virus and Citrus exocortis viroid.