Vector transmission of parsley yellow leaf curl virus by the leafhopper Austroagallia sinuata

This study investigates the transmission potential of parsley yellow leaf curl virus (PYLCV; family Geminiviridae) by Austroagallia sp. leafhopper commonly found in vegetable farms in Kerman province, Iran. Accordingly, following two days of acquisition access period, nymphs from the non-viruliferous colony of Austroagallia sp. successfully transmitted the virus from PYLCV infected parsley to healthy seedlings after seven days from the start of feeding. On the basis of male genitalia morphology, the species of the leafhopper, was identi�ed as A. sinuata. To our knowledge, this is the �rst report of a plant virus transmission by a species of Austroagallia in the world.


Main Text
The family Geminiviridae is a large group of plant-infecting viruses with unique particle morphology consisting of two jointed incomplete icosahedral capsids (twined particles) about 22x38 nm in size [1][2][3].The genome of geminiviruses consists of one or two circular single-stranded DNA molecules of 2.5-2.5 kb in size.Except for some members of the genus Begomovirus which have bipartite genome, members of other genera have monopartite genome [4].Based on the host range, type of the insect vector and genome organization, geminiviruses are classi ed into 14 genera [5].These viruses infect a wide range of plants belonging to different families and cause damage to crops, vegetables, ber crops and fruit trees [6].
Members of different genera of the family Geminiviridae are transmitted in nature by a relatively wide range of insect vectors, such as leafhoppers, treehoppers, aphids and white ies via a persistent and circulating manner [7].However, insect vector has not yet been reported for some geminiviruses [5].
Among insect vectors of geminiviruses, leafhoppers (members of the family Cicadellidae) are a major vector [5,7].Damages caused by members of this family are due to feeding on plant sap and reducing the yield of crops, as well as the transmission of pathogens including viruses [8], phytoplasmas [9] and a number of other pathogenic prokaryotes [10,11].
PYLCV was reported for the rst time in 2020 from Bagher-Abad (Kerman province) located in the southeast of Iran, inducing leaf curling, marginal leaf yellowing, dwar sm and reduced leaf size in parsley (Fig. 1a).The complete genome of the virus shared < 66% genome-wide pairwise identity to all known geminiviruses.Although the virion sense encoded proteins are most similar to those of becurtoviruses and curtoviruses, the complementary sense encoded proteins are most similar to those of begomoviruses [12].According to the last report of the International Committee on Taxonomy of Viruses (ICTV), while identity of four viruses, i.e., PYLCV, Limeum africanum associated virus, common bean curly stunt virus and Polygala garcinii associated virus have been con rmed as the members of the family Geminiviridae, but due to their genomic characteristics, they have not yet been classi ed in any of the current 14 genera [5].
The yield of leafy vegetables in Iran is affected by various viruses, such as geminiviruses and nanoviruses [12][13][14][15].Nevertheless, in comparison to well-known crops, viruses infecting leafy vegetable have not yet been adequately studied.For example, many biological features of PYLCV such as its vector and host range remain unknown.For this reason, in this research, the insect transmission of this virus has been investigated.In geminiviruses, the coat protein (CP), in addition to encapsidate the virus particles, may has more additional functions, such as the transmission of the virus by the vector [7].
Based on previous studies, the CP of PYLCV shared less than 56% amino acid pairwise identity with those encoded by all other known geminiviruses such as becurtoviruses and curtoviruses.Species within these two genera in Iran, namely Beet curly top Iran virus [16] and Beet curly top virus [17] as well as three turncurtoviruses [18,19] are naturally transmitted by C. haematoceps.Hence, it was predicted that PYLCV may be also transmitted by a leafhopper such as C. haematoceps [12].On the other hand, another frequently found leafhopper in vegetable-growing farms is A. sinuata [20].Therefore, in this research, in order to identify the vector of PYLCV, the role of two dominant leafhoppers in the transmission of this virus in the leafy vegetable farms of Kerman province was studied.
Leafhoppers were collected from the leafy vegetable farms of South Rudbar-e-Jonoob located 320 km south of Kerman province using an insect sweep net and were transferred to greenhouse under cold conditions using an ice box.Subsequently, adult insects from two genera, Circulifer and Austroagallia, were separated using external morphology [21][22][23][24][25] and caged on healthy parsley seedlings using an aspirator and transparent cylinder sealed at one end with cotton mesh.Leafhoppers were kept under insect-proof conditions in greenhouse at ~ 25 ºC.After 20 days from start of feeding by the leafhoppers, total DNA was extracted from parsley plants using the CTAB method [26] and checked by PCR assay using speci c primer pair PYLCV-F-446/PYLCV-R-1275 [12] to assure their freedom from known leafhopper-borne geminiviruses of vegetables and crops found in Iran, namely PYLCV [12], beet curly top Iran virus (BCTIV) [27], beet curly top virus (BCTV) [28] and three turncurtoviruses [18,19].Non-viruliferous leafhoppers were used for the next stage of experiment.
To prepare PYLCV infected parsley seedlings for the virus acquisition by leafhoppers, constructed infectious clone of PYLCV was used for agroinoculation of plants as previously described [12] and the PYLCV infection of agroinoculated plants was con rmed using appearance of viral symptoms and PCR assay after 15 dpi.Subsequently, different nymphal instars from both groups of non-viruliferous leafhoppers (Circulifer and Austroagallia; two nymphs per plant) were separately caged on symptomatic PYLCV infected parsley plants using transparent cylinder to allow for acquisition of the virus under greenhouse conditions as previously mentioned.After two days from the start of feeding, leafhoppers from both genera were separately transferred to the healthy parsley seedlings at the 4-5 leaf stage using an aspirator for inoculation of the virus.After 20 days from the start of feeding, total DNA were extracted from inoculated parsley plants nd the PYLCV infection of plants was checked using appearance of the viral symptoms and PCR assay.
Based on the results of the last stage of experiment, about 20% inoculated of parsley plants by the leafhoppers, Austroagallia sp., showed typical PYLCV symptoms including marginal leaf yellowing and dwar ng (Fig. 1b).The PCR assay of symptomatic parsley plants resulted in expected 830 bp amplicon in 1% agarose gel (Fig. 2) followed by sequencing of PCR products which con rmed the PYLCV infection of PCR-positive samples.Whereas no symptom appeared in parsley plants that were fed by the leafhoppers, Circulifer sp. and no amplicon was detected in PCR assay.Hence, based on these results, Austroagallia sp. was identi ed to be the vector of PYLCV.Until now, two species for the genus Austroagallia have been documented to exist in Iran [24].To identify the species of the viruliferous leafhopper (Austroagallia), one adult male and one adult female of Austroagallia were cage on individual parsley seedlings at the 4-5 leaf stage using an aspirator for inoculation access period in eight replicates.From one week after the start of feeding, the PYLCV infection of the parsley plants was detected for two replicates (out of eight) using appearance of the viral symptoms and then the PCR assay as previously mentioned.Subsequently, male genitalia of the viruliferous Austroagallia was extracted from leafhoppers of two PCR positive replicates as previously described [29] and compared with those of Austroagallia species such as A. sinuata (Fig. 3) [25].Based on the obtained results, the leafhopper A. sinuata (family Cicadellidae) was able to transmit PYLCV from infected parsley to healthy parsley seedlings under greenhouse conditions.
Up to now, members of ve genera in the family Geminiviridae including Becurtovirus, Curtovirus, Turncurtovirus, Mastrevirus and Mulcrilevirus (out of 14 genera) are transmitted by leafhoppers [5,7].Also, until this research, A. sinuata was only reported as the vector of some phytoplasma and spiroplasma from Iran and some parts of the world, respectively [30,31].This leafhopper has been reported as a vector of phytoplasma, namely Candidatus phytoplasma aurantifolia, in Iran from common zinnia (Zinnia elegans) [31].Therefore, according to the results of this study and based on the available information, this is the rst report of the transmission of a plant virus by one species of leafhopper Austroagallia in the world.
One of the criteria used by International Committee on Taxonomy of Viruses (ICTV) to classify geminiviruses into different genera, is the type of insect vector [7].Currently, despite PYLCV has been accepted as a distinct geminivirus by ICTV [5], due to its unique genome characteristics and the unknown biological features, such as insect vector, this virus has not yet been classi ed in the 14 genera of the family Geminiviridae.The identi cation of A. sinuata as the vector of PYLCV sheds light on one of the unknown aspects of this virus, and considering the fundamental role of the vector in the classi cation of geminiviruses, the results of this study can certainly be effective in determining its taxonomic position.

Declarations Figures
of naturally infected parsley with parsley yellow leaf curl virus showing leaf curling and marginal leaf yellowing collected in parsley farms in Zafar-Abad, Fars province (a), and those of infected parsley three weeks after feeding of viruliferous Austroagallia sinuata from healthy parsley plants (b) under greenhouse conditions.