Identification of Phytoplasmas Representing Multiple New Genetic Lineages from Phloem-Feeding Leafhoppers Highlights the Diversity of Phytoplasmas and Their Potential Vectors.

Wei Wei,Valeria Trivellone,Christopher H Dietrich,Algirdas Ivanauskas,Kristi D Bottner-Parker,Yan Zhao

doi:10.3390/pathogens10030352

Abstract

Phytoplasmas are obligate transkingdom bacterial parasites that infect a variety of plant species and replicate in phloem-feeding insects in the order Hemiptera, mainly leafhoppers (Cicadellidae). The insect capacity in acquisition, transmission, survival, and host range directly determines the epidemiology of phytoplasmas. However, due to the difficulty of insect sampling and the lack of follow-up transmission trials, the confirmed phytoplasma insect hosts are still limited compared with the identified plant hosts. Recently, quantitative polymerase chain reaction (qPCR)-based quick screening of 227 leafhoppers collected in natural habitats unveiled the presence of previously unknown phytoplasmas in six samples. In the present study, 76 leafhoppers, including the six prescreened positive samples, were further examined to identify and characterize the phytoplasma strains by semi-nested PCR. A total of ten phytoplasma strains were identified in leafhoppers from four countries including South Africa, Kyrgyzstan, Australia, and China. Based on virtual restriction fragment length polymorphism (RFLP) analysis, these ten phytoplasma strains were classified into four distinct ribosomal (16Sr) groups (16SrI, 16SrIII, 16SrXIV, and 16SrXV), representing five new subgroups (16SrI-AO, 16SrXIV-D, 16SrXIV-E, 16SrXIV-F, and 16SrXV-C). The results strongly suggest that the newly identified phytoplasma strains not only represent new genetic subgroup lineages, but also extend previously undiscovered geographical distributions. In addition, ten phytoplasma-harboring leafhoppers belonged to seven known leafhopper species, none of which were previously reported insect vectors of phytoplasmas. The findings from this study provide fresh insight into genetic diversity, geographical distribution, and insect host range of phytoplasmas. Further transmission trials and screening of new potential host plants and weed reservoirs in areas adjacent to collection sites of phytoplasma harboring leafhoppers will contribute to a better understanding of phytoplasma transmission and epidemiology.

Highlights

Phytoplasmas are a large group of phloem-restricted, cell wall-less bacteria that infect nearly a thousand plant species and cause serious economic loss worldwide
The presence of phytoplasma in 76 leafhopper samples collected in natural habitats was investigated by semi-nested polymerase chain reaction (PCR) amplification employing phytoplasma specific primers
Amplicons of around 1.5 kb were obtained from ten single leafhopper specimens (Table 1, Supplementary Figure S1 and Table S1); among them, six specimens were phytoplasma-positive in quantitative polymerase chain reaction (qPCR)-based prescreening [24]

Summary

Introduction

Phytoplasmas are a large group of phloem-restricted, cell wall-less bacteria that infect nearly a thousand plant species and cause serious economic loss worldwide. Phytoplasmas are transmitted by phloem sap feeding insect vectors, mainly leafhoppers, in a persistent-propagative manner [1,2,3]. Phloem feeding insects obtain phytoplasmas from diseased plants during feeding, and the phytoplasmas penetrate the intestinal wall of the insect (the first barrier) and circulate in the hemolymph. The phytoplasmas further enter the salivary glands (the second barrier) and multiply there. At this point, the insects become vectors. Once they feed on healthy plants, the plants become infected [1,2,3]

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