Detection of \u2018Candidatus Phytoplasma solani\u2019 in roots from Bois noir symptomatic and recovered grapevines

Lucia Landi,Sergio Murolo,Gianfranco Romanazzi

doi:10.1038/s41598-018-38135-9

Abstract

‘Candidatus Phytoplasma solani’ is the causal agent of Bois noir (BN) in grapevine (Vitis vinifera). It is usually detected in leaves, where typical disease symptoms are seen. However, little information is available on the presence of this phytoplasma in grapevine roots. Here, we investigated ‘Ca. P. solani’ in roots collected from 28 symptomatic, 27 recovered and eight asymptomatic grapevine plants. Protocols based on high-resolution melting (HRM) combined with real-time quantitative PCR (qPCR-HRM) and nested-qPCR-HRM were developed to identify ‘Ca. P. solani’ tuf-type variants with single nucleotide polymorphisms. In all, 21.4% of roots from symptomatic plants were positive to ‘Ca. P. solani’ using qPCR-HRM, and 60.7% with nested-qPCR HRM. Also, 7.4% of roots from recovered plants were positive using qPCR-HRM, which reached 44.4% using nested-qPCR HRM. These analyses identified tuf-type b1 on 88.2% of the positive samples from symptomatic grapevines, and 66.6% from recovered grapevines, with all other samples identified as tuf-type a. This study reports the presence of ‘Ca. P. solani’ in the roots of both symptomatic and recovered grapevines. These qPCR-HRM and nested-qPCR-HRM protocols can be applied to increase the sensitivity of detection of, and to simplify and speed up the screening for, ‘Ca. P. solani’ tuf-types.

Highlights

Grapevine yellows are diseases that can have detrimental effects upon grapevine yields, in terms of both quantity and quality[1,2]
It has been demonstrated that Candidatus Phytoplasma australiense’ (16SrXII-B) and Tomato big bud phytoplasma (16SrII-D), associated with Australian Grapevine Yellows[10], and ‘Ca. P. solani’[11], have been detected in trunk, cordon, shoots, and roots of phytoplasma-affected grapevines
Hren et al.[16] reported weak amplicons associated with the presence of Flavescence dorée (FD) phytoplasma in one out of six Barbera grapevines that had recovered from FD disease, another important grapevine yellows disease in Europe

Summary

Introduction

Grapevine yellows are diseases that can have detrimental effects upon grapevine yields, in terms of both quantity and quality[1,2]. The optimal period for diagnosis of ‘Ca. P. solani’ in grapevine leaves in the northern hemisphere is generally from June to September. This is prior to harvest, which for the Chardonnay cultivar is expected from the mid of August to the beginning of September[2]. An intriguing aspect of the epidemiology of BN is the process of ‘recovery’, which is the spontaneous disappearance of BN symptoms from previously symptomatic plants[13,14] In such recovered grapevines, attempts to detect phytoplasma in the canopy have usually failed[15]. Correspondence and requests for materials should be addressed to G.R. (email: g.romanazzi@ univpm.it) www.nature.com/scientificreports/

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