Sunn hemp, a major source-plant of the phytoplasma associated with huanglongbing symptoms of sweet orange in São Paulo State, Brazil

L. F. Bianco,R. S. Toloy,E. W. Kitajima,D. A.B. Colletti,E. C. Martins,N. A. Wulff,J. M. Bové,D. C. Teixeira

doi:10.5070/c421026956

Abstract

In Sao Paulo State (SPS), sweet orange (Citrus sinensis) trees with huanglongbing (HLB) symptoms are infected with Candidatus (Ca.) Liberibacter (L.) asiaticus (Las) or Ca. L. americanus (Lam). However, in 2007, 3 years after HLB was first reported in SPS, some trees with characteristic HLB symptoms were found free of liberibacters, but infected with a phytoplasma of 16Sr group IX. This phytoplasma was further characterized by PCR amplification of ribosomal protein genes rpsC-rplV-rpsS and amplicon sequencing. A qPCR test to detect the phytoplasma in plants and insects was also developed on the basis of the ribosomal protein genes. The phytoplasma was transmitted from citrus-to-citrus by grafting. The 16Sr group IX phytoplasma associated with HLB symptoms in sweet orange in SPS and characterized by the above techniques was named “HLB-phytoplasma”. Although the HLB-phytoplasma is widely distributed in many municipalities of central, northern, and northwestern SPS, the number of HLB-phytoplasma-infected trees in each municipality is very small. Experiments have been undertaken to identify the origin of the HLB-phytoplasma and the source of inoculum on which a putative insect vector could become infected with the HLB-phytoplasma. In SPS, sunn hemp (Crotalaria juncea L.) is a major, widely distributed cover crop. A 16Sr group IX phytoplasma was detected in sunn hemp plants with witches’ broom and virescence symptoms, and was shown to have 16Sr DNA sequences and ribosomal protein gene sequences with 100% identity to the corresponding sequences of the sweet orange HLB-phytoplasma. Transmission electron microscopy revealed the presence of phytoplasma cells in the phloem sieve tubes of infected C. juncea stalks. These results were taken as evidence that the sunn hemp phytoplasma and the sweet orange HLB-phytoplasma were identical. Scaphytopius marginelineatus, a leafhopper frequently found in sweet orange orchards, was shown to acquire the HLB-phytoplasma efficiently from affected sunn hemp plants, but acquisition from, and transmission rates to, sweet orange were very low. On the whole, these data suggest that (i) sunn hemp is a major source of inoculum of the HLB-phytoplasma, (ii) S. marginelineatus becomes infected on sunn hemp and transmits the phytoplasma to sweet orange, and (iii) transmission from sweet orange to sweet orange occurs only rarely, if at all. 16Sr group IX phytoplasmas, very closely related to the SPS HLB-phytoplasma, have also been detected in citrus in Minas Gerais and Bahia states (Brazil) and Mexico.

Highlights

Before 2004, only 2 species of Candidatus (Ca.) Liberibacter (L.) were known to be associated with citrus huanglongbing (HLB): Ca
Most of the affected trees are distributed randomly and, in 80% of cases, the minimum distances between affected trees are higher than 100 m (Teixeira et al 2008). These results suggest that primary infection of citrus trees with the 16Sr group IX phytoplasma by the putative leafhopper vector, possibly S. marginelineatus, is a rare event, and that secondary infections from citrus-to-citrus by D. citri, the Asian HLB psyllid vector, do not occur
2 young, symptomatic sweet orange field trees from São Paulo State (SPS) were detected, one from Potirendaba MU and one from Parisi MU; they were transferred in 2008 from the orchards to 100 L containers inside the Fundecitrus greenhouse in Araraquara, SPS. The phytoplasmas from these 2 sweet orange trees were confirmed as the HLB-phytoplasma by nested PCR amplification of 16Sr DNA with primers P1/P7 followed by primers fU5/rU3, and sequencing of the nested amplicon (Table 1, PCR technique “a1”)

Summary

Introduction

Before 2004, only 2 species of Candidatus (Ca.) Liberibacter (L.) were known to be associated with citrus huanglongbing (HLB): Ca. L. africanus (Laf) in Africa and Ca. L. asiaticus (Las) in Asia (Bové 2006). In 2004, HLB was reported for the first time from America and, more precisely, from central São Paulo State (SPS), Brazil. Two Ca. L. species were identified and could be detected by specific PCR techniques (Teixeira, Ayres, et al 2005; Teixeira, Saillard, et al 2005). Most trees were infected with a new liberibacter species, Ca. L. americanus (Lam), and only very few trees carried Las, the known Asian species. Over the years, the proportion of Las-infected trees has increased greatly, while Lam-infected trees have become difficult to find (Lopes et al 2009). Since 2005, when Florida, USA, reported for the first time the iocv_journalcitruspathology_26956

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