Preferred name: 'Candidatus Phytoplasma aurantifolia'
Authority: Zreik, Bové & Garnier
Taxonomic position: Bacteria: Tenericutes: Mollicutes: Acholeplasmatales: Acholeplasmataceae
Other scientific names: Lime witches' broom phytoplasma, Phytoplasma aurantifolia Zreik, Bové & Garnier
Common names in English: Oman witches' broom disease, WBDL, witches' broom of lime
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Notes on taxonomy and nomenclature EU Categorization: A1 Quarantine pest (Annex II A)
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EPPO Code: PHYPAF

The main natural host of lime witches' broom phytoplasma is Citrus aurantiifolia, the small-fruited acid lime which is grown mainly in India, China, Arabian Peninsula, Mexico, Southern Iran, the West Indies, Egypt, and the tropical Americas. 

As shown in the host list below, some other Citrus species can also be a natural host of ‘Ca. P. aurantifolia’ (Hemmati et al., 2021a). In Iran, the disease is not frequent on Citrus other than C. aurantiifolia and Bakraee (a natural hybrid of C. reticulata commonly used as a rootstock in Iran), and its occurrence appears to depend on the susceptibility of the rootstock, the vector populations, disease pressure in the infected region (% incidence), and the presence of infected lime trees in the vicinity (Salehi et al., 2022). 

In experiments, the phytoplasma has been graft-transmitted to several Citrus species, including Citroncirus webberi, Citrus macrophylla and C. trifoliata, but at a lower frequency and with slow symptom development (Garnier et al., 1991, Hassanzadeh et al., 2019; Azadvar et al., 2023). Persian lime (Citrus × latifolia) is known as the most resistant lime cultivar to 'Ca. P. aurantifolia' (Hassanzadeh et al., 2019). Dodder (Cuscuta) has been used to transmit the phytoplasma to Catharanthus roseus, a widely used indicator plant for phytoplasmas.

Host list: Citrus hybrids, Citrus medica, Citrus x aurantiifolia, Citrus x aurantium var. paradisi, Citrus x aurantium var. sinensis, Citrus x limon var. limetta, Citrus x limon var. limettioides, Citrus x limon, Citrus x limonia var. jambhiri, Citrus x tangelo, x Citrofortunella floridana

The witches' broom disease of lime associated with 'Ca. P. aurantifolia' was reported in a few countries in Asia including Oman (Bové, 1986), the United Arab Emirates (Garnier et al., 1991), and Iran (Bové et al., 2000).

An asymptomatic infection of lime in Brazil was observed and mentioned to belong to the subgroup II-C by Silva et al. (2014). However, Al-Subhi et al. (2021) stated that the sequences of the imp, 16SrRNA, and tuf genes from some phytoplasma isolates from Oman, Iran, Brazil, the United Arab Emirates and Saudi Arabia were identical and grouped with 16SrII-B ('Ca. P. aurantifolia') in phylogenetic analysis.

Asia: Iran, Oman, United Arab Emirates
South America: Brazil (Minas Gerais, Santa Catarina, Sao Paulo)

Lime witches' broom is the first well-characterized phytoplasma disease of citrus. It is an economically important and destructive disease and a significant limiting factor on acid lime production in the Middle East and threatens its production globally. Hishimonus phycitis is known as the main vector of the associated phytoplasma to healthy mature lime trees and seedlings (Bagheri et al., 2009; Hemmati et al., 2020). The Asian psyllid Diaphorina citri was also able to experimentally transmit the phytoplasma at a lower efficiency in a no-choice assay (Queiroz et al., 2016), and no report is available regarding the natural transmission. To date, seed transmission of 'Ca. Phytoplasma aurantifolia' has not been proven (Faghihi et al., 2011).

Symptoms

On lime, witches' broom symptoms develop over the whole tree, accompanied by the production of small-sized leaves on proliferating shoots that tend to be yellowish-green, and reduced flower and fruit production. These are found in clusters on the branches of infected acid lime trees. The symptoms develop on branches gradually and the trees become unproductive until the whole tree collapses within 4-8 years of the first appearance of symptoms (Al-Ghaithi et al., 2017). In some cases, no symptoms have been observed in infected trees (Silva et al., 2014, Al-Subhi et al., 2021).

Morphology

The presence of 200-800 nm-sized phytoplasma cells that are pleomorphic can be easily observed through electron microscopy in sieve tubes (Garnier et al., 1991).

Detection and inspection methods

Visual symptoms are important for diagnosis in symptomatic plants (above-mentioned symptoms). Although 'Ca. P. aurantifolia' is usually symptomatic, it may be present in the absence of symptoms due to its long incubation period, or the reaction of some cultivars like in Brazil (Silva et al., 2014). The phytoplasma has been detected in infected trees through the use of monoclonal antibodies, PCR, and real-time PCR, by using specific primers (Al-Subhi et al., 2021; Hemmati et al., 2021b). Mexican lime and periwinkle can be used as indicators in graft and biological transmission assays. In laboratories in the EPPO region, these methods are less used currently because of the lack of validation data, and detection using PCR followed by identification by sequence analysis is recommended, as described in the EPPO diagnostic protocol PM 7/133 (1) Generic detection of phytoplasmas (EPPO, 2018).

Lime witches' broom disease has spread naturally in Asian countries via its vector. Globally, there is a higher chance to distribute the disease with infected plants for planting. The emergence of witches' broom disease of lime in Oman, a region that has had a longstanding tradition of cultivating limes, raises the plausible assumption that the disease may have been imported from external sources. The entry of ‘Ca. Phytoplasma aurantifolia’ into the EPPO region could happen via infected plants for planting or vectors. However, the import of Citrus plants for planting from countries where the pest occurs is prohibited in the EU (EU, 2023). The prohibition of import is likely in other Citrus-producing countries in the EPPO region.

Entry of the phytoplasma with infected adults and nymphs of the leafhopper vector Hishimonus phycitis is unlikely because they move and leap away from plants when disturbed, therefore, it is highly improbable that these mobile stages would remain on host plant materials as it is handled along a pathway. Eggs of H. phycitis could be present on the plants, but to date, it remains unknown if eggs can carry ‘Ca. P. aurantifolia’ because it remains unknown if there is transovarial transmission of the phytoplasma to eggs.

Economic impact

Symptoms are severe and rapidly affect the whole tree, inhibiting flowering, and fruiting. Over 50% of the cultivated area of lime has been lost in Oman since it was reported in the 1970s and hundreds of thousands of lime trees have been destroyed in Iran due to witches’ broom disease of lime. The disease occurs widely in the coastal region of the Oman Sea. The potential impact of the disease is limited to those countries that grow acid lime commercially (see Phytosanitary risk). This disease is deemed significant enough to warrant the consideration of implementing a programme to completely eliminate it.

Control

Given the lack of viable solutions for treating plants infected with phytoplasma, the primary focus is on implementing preventative measures, including agronomic techniques and vector management, to minimize the spread and impact of the disease. To effectively manage witches’ broom disease of lime, it is strongly advised to utilize disease-free seedlings and planting materials and shield the nursery with a net that protects against insects. Routinely applying systemic insecticides is another strategy for controlling the vectors. Newly infected trees can be safeguarded by removing symptomatic branches, while highly infected trees should be eliminated to prevent further spread of the disease. Opting for resistant or tolerant species such as Persian lime and controlling weeds (which may serve as a reservoir for phytoplasmas) are additional measures that are highly recommended (Azadvar et al., 2023).

Phytosanitary risk

Acid lime (C. aurantifolia), the main host of 'Ca. P. aurantifolia', is mostly not grown commercially in the EU because of ecoclimatic limitations (EFSA, 2017), although there is a small commercial cultivation in limited areas, such as Sicily in Italy (Gusella et al., 2021). Acid lime is also produced commercially, on a smaller scale than other citrus, in the warmest parts of the EPPO region, for example in Morocco and Israel (Sammama et al., 2016; EFSA, 2021a).

Other citruses, especially orange (C. sinensis), grapefruit (C. paradisi), as well as mandarins and clementines (C. reticulata), are the most economically important Citrus crops in the Mediterranean area that can be infected (Hemmati et al., 2021a, Salehi et al., 2022). So far, it has been observed that the incidence of infection of ‘Ca. P. aurantifolia’ on such crops is relatively infrequent in nature (Salehi et al., 2022). In Iran, particular combinations of factors were identified as being contributory to such infection (Salehi et al., 2022; see under the Hosts section), but the same conditions are currently not met within the EPPO region.

In order to prevent the introduction of the disease to the EPPO region, the importation of Citrus tree plants for planting, originating from areas where the disease is prevalent, can be prohibited (CABI/EPPO, 1997), as is already the case in many EPPO countries. Alternatively, measures such as pest-free areas may be appropriate, as is the case for similar pests, as well as pest-free production sites or pest-free places of production with conditions also ensuring the absence of vectors. Healthy planting material of Citrus can be produced in the framework of a certification scheme. A pest survey card was prepared by the European Food Safety Authority (EFSA, 2021b) to assist EU Member States in planning their annual survey activities for ‘Ca. Phytoplasma aurantifolia’ and its vector H. phycitis.

Al‐Ghaithi AG, Al‐Sadi AM, Al‐Hammadi MS, Al‐Shariqi RM, Al‐Yahyai RA, Al‐Mahmooli IH, Carvalho CM, Elliot SL & Hogenhout SA (2017) Expression of phytoplasma‐induced witches’ broom disease symptoms in acid lime (Citrus aurantifolia) trees are affected by climatic conditions. Plant Pathology 66(8), 1380-1388.

Al-Subhi AM, Al-Sadi AM, Al-Yahyai RA, Chen Y, Mathers T, Orlovskis Z, Moro G, Mugford S, Al-Hashmi KS & Hogenhout SA (2021) Witches’ broom disease of lime contributes to phytoplasma epidemics and attracts insect vectors. Plant Disease 105(9), 2637-2648.

Azadvar M, Esmaeilzadeh-Hosseini SA, Salehi M, Al-Subhi AM, Hemmati C, Al-Ghaithi A & Faghihi MM (2023) Updates on phytoplasma diseases associated with Citrus crops in Asia. In Phytoplasma Diseases of Major Crops, Trees, and Weeds (eds Tiwari A, Caglayan K, Hoat T, Al-Subhi A, Nejat N, Reddy G) pp. 265-281. Academic Press.

Bagheri AN, Salehi M, Faghihi MM, Samavi S & Sadeghi A (2009) Transmission of 'Candidatus Phytoplasma aurantifolia' to Mexican lime by the leafhopper Hishimonus phycitis in Iran. Journal of Plant Pathology 91(4), 105.

Bové J (1986) Outbreaks and new records. Oman. Witches' broom disease of lime. FAO Plant Protection Bulletin 34(4), 217-218.

Bové JM, Danet JL, Bananej K, Hassanzadeh N, Taghizadeh M, Salehi M & Garnier M (2000) Witches’ broom disease of lime (WBDL) in Iran. International Organization of Citrus Virologists Conference Proceedings (1957-2010) 14(14), 207-212.

CABI/EPPO (1997) Lime witches' broom phytoplasma. Data Sheets on Quarantine Pests. pp. 1022-1024. In Quarantine Pests for Europe (2^nd edition). CABI, Wallingford (GB).

EFSA (2017) EFSA Panel on Plant Health (PLH), Jeger M, Bragard C, Candresse T, Chatzivassiliou E, Dehnen‐Schmutz K, Gilioli G, Gregoire JC, Jaques Miret JA, MacLeod A & Navarro MN. Pest categorization of Witches' broom disease of lime (Citrus aurantifolia) phytoplasma. EFSA Journal 15(10), 05027. 22 p. https://doi.org/10.2903/j.efsa.2017.5027

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EFSA (2021b) Pest survey card on ‘Candidatus Phytoplasma aurantifolia’ and its vector Hishimonus phycitis. EFSA supporting publication 2021:EN-7026. Available online: https://arcg.is/19SK8v. Last updated: 30 November 2021. https://doi.org/10.2903/sp.efsa.2021.EN-70

EPPO (2018) EPPO Standards PM 7/61 (1) Diagnostics. Generic detection of phytoplasmas. EPPO Bulletin, 48(3), 414-424.

EU (2023) Commission Implementing Regulation (EU) 2019/2072 of 28 November 2019 establishing uniform conditions for the implementation of Regulation (EU) 2016/2031 of the European Parliament and the Council, as regards protective measures against pests of plants, and repealing Commission Regulation (EC) No 690/2008 and amending Commission Implementing Regulation (EU) 2018/2019. Consolidated version 32019R2072, 11/01/2023. https://eur-lex.europa.eu/legal-content/EN/TXT/?uri=CELEX%3A32019R2072 [Accessed on 8 May 2023].

Faghihi MM, Bagheri AN, Bahrami HR, Hasanzadeh H, Rezazadeh R, Siampour M, Samavi S, Salehi M & Izadpanah K (2011) Witches'-broom disease of lime affects seed germination and seedling growth but is not seed transmissible. Plant Disease 95(4), 419-22.

Garnier M, Zreik L & Bové JM (1991) Witches' broom, a lethal mycoplasmal disease of lime trees in the sultanate of Oman and the United Arab Emirates. Plant Disease 75, 546-551.

Gusella G, Fiorenza A, Aiello D & Polizzi G (2021) Unusual stylar-end breakdown and sour rot on key lime (Citrus aurantiifolia) in pre-harvest condition in Italy. Plants 10, 989. 7 pp.

Hassanzadeh Khankahdani HH, Bahrami HR, Faghihi MM & Bagheri A (2019) Reaction of some commercial citrus species and Iranian lime biotypes to witches' broom disease of lime. Crop Protection 122, 23-29.

Hemmati C, Askari Seyahooei M, Nikooei M, Modarees Najafabadi SS, Goodarzi A, Amiri Mazraie M & Faghihi MM (2020) Vector transmission of lime witches' broom phytoplasma to Mexican lime seedlings under greenhouse condition. Journal of Crop Protection 9(2), 209-215.

Hemmati C, Nikooei M, Al-Subhi AM & Al-Sadi AM (2021a) History and current status of phytoplasma diseases in the Middle East. Biology 10(3), 226.

Hemmati C, Nikooei M & Al-Sadi AM (2021b) Five decades of research on phytoplasma-induced witches' broom diseases. CABI Reviews 16(002), 1-6.

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Salehi M, Faghihi MM, Salehi E, Ebadi N & Bertaccini A (2022) Molecular and biological characterization of ‘Candidatus Phytoplasma aurantifolia’ strains infecting commercial citrus species in Iran. Archives of Phytopathology and Plant Protection 55(16), 1879-1898.

Sammama A, Elyahyaoui O, Kerrouri S, Bouabid B, Ould AL, Al Rhorfi L & Bengueddour R (2016) Qualitative study in vitro fruit and epicarpes Citrus limetta Risso, Citrus limon Burm. and Citrus aurantiifolia (Christm.) Swingle Gharb of Morocco. Journal of Advances in Biology 9(3), 1911-1921.

Silva FN, Queiroz RB, Souza AN, Al-Sadi AM, Siqueira DL, Elliot SL & Carvalho CM (2014) First report of a 16SrII-C phytoplasma associated with asymptomatic acid lime (Citrus aurantifolia) in Brazil. Plant Disease 98(11), 1577.

Zreik L, Carle P, Bové JM & Garnier M (1995) Characterization of the mycoplasmalike organism associated with witches'-broom disease of lime and proposition of a Candidatus taxon for the organism, 'Candidatus Phytoplasma aurantifolia'. International Journal of Systematic Bacteriology 45(3), 449-453.

This datasheet was extensively revised in 2023 by Chamran Hemmati and Mehrnoosh Nikooei (University of Hormozgan, Iran). Their valuable contribution is gratefully acknowledged.

EPPO (2024) 'Candidatus Phytoplasma aurantifolia'. EPPO datasheets on pests recommended for regulation. https://gd.eppo.int (accessed 2024-04-27)

This datasheet was first published in 1997 in the second edition of 'Quarantine Pests for Europe', and revised in 2023. It is now maintained in an electronic format in the EPPO Global Database. The sections on 'Identity', ‘Hosts’, and 'Geographical distribution' are automatically updated from the database. For other sections, the date of last revision is indicated on the right.

CABI/EPPO (1997) Quarantine Pests for Europe (2^nd edition). CABI, Wallingford (GB).