Grapevine Planting Research Articles

Hot water treatment of Vitis sp. for Xylella fastidiosa

Following a request from the European Commission, the EFSA Panel on Plant Health (PLH) reviewed Italian technical guidelines and the ANSES (Agence nationale de sécurité sanitaire de l'alimentation, de l'environnement et du travail) opinion on the use of hot water treatment (HWT) on Vitis sp. planting material, assessing its efficacy in the elimination of the xylem-invading bacterial pathogen, Xylella fastidiosa. HWT is a robust and reliable technique used to destroy life stages of pests (insects, nematodes) and to inactivate pathogens (phytoplasma, bacteria, fungi) in dormant plant propagation materials (grapevine and other crops). An effective HWT sanitizes the planting material without affecting plant survival and development. For grapevine, HWT to eliminate the Grapevine flavescence dorée phytoplasma (FD) from planting materials is among the special requirements for the introduction and movement of Vitis sp. to protected zones in the EU. The conditions of 50°C for 45 min, prescribed and recommended to sanitize grapevine planting material against FD, are considered by the Panel to be also effective against X. fastidiosa and its subspecies. Despite uncertainties on variable thermotolerances of the bacteria, a HWT treatment of 50°C for 45 minutes can effectively account for different thermotolerances. It should be noted that the quality of the HWT is subject to the proper application of the operating procedures to guarantee vigorous growth and pathogen freedom of planting material.

Genetic relatedness and recombination analysis of Allorhizobium vitis strains associated with grapevine crown gall outbreaks in Europe.

To analyse genetic diversity and epidemiological relationships among 54 strains of Allorhizobium vitis isolated in Europe during an 8-year period and to assess the relative contribution of mutation and recombination in shaping their diversity. By using random amplified polymorphic DNA (RAPD) PCR, strains studied were distributed into 12 genetic groups. Sequence analysis of dnaK, gyrB and recA housekeeping genes was employed to characterize a representative subcollection of 28 strains. A total of 15 different haplotypes were found. Nucleotide sequence analysis suggested the presence of recombination events in A. vitis, particularly affecting dnaK locus. Although prevalence of mutation over recombination was found, impact of recombination was about two times greater than mutation in the evolution of the housekeeping genes analysed. The RAPD analysis indicated high degree of genetic diversity among the strains. However, the most abundant RAPD group was composed of 35 strains, which could lead to the conclusion that they share a common origin and were distributed by the movement of infected grapevine planting material as a most common way of crossing long distances. Furthermore, it seems that recombination is acting as an important driving force in the evolution of A. vitis. As no substantial evidence of recombination was detected within recA gene fragment, this phylogenetic marker could be reliable to characterize phylogenetic relationships among A. vitis strains. We demonstrated clear epidemiological relationship between majority of strains studied, suggesting a need for more stringent phytosanitary measures in international trade. Moreover, this is the first study to report recombination in A. vitis.

Development of a monoclonal antibody TAS-ELISA assay for detection of Phaeomoniella chlamydospora

Summary. Phaeomoniella chlamydospora is an important tracheomycotic fungus involved in different syndromes of the esca disease complex affecting young and mature grapevine plants. Although grapevine planting material is frequently infected by this pathogen, no regular screening to detect the fungus is carried out, mainly due to the lack of fast, sensitive and affordable methods for large-scale application. A monoclonal antibody-based triple antibody sandwich (TAS) enzyme-linked immunosorbent assay (ELISA) was developed to specifically detect Pa. chlamydospora from grapevine wood tissues. This assay could be especially useful for large-scale application in nurseries, to ensure Pa. chlamydospora-free grapevine stocks, and thus contributing to the production of healthy propagation material.

Detection of black-foot disease pathogens in the grapevine nursery propagation process in Spain

Two commercial nurseries located in Comunidad Valenciana region (central-eastern Spain) were sampled in 2010 to evaluate whether the grapevine nursery propagation process could be a source of black-foot disease pathogens. Samples were taken from four sources of the propagation process: pre-grafting hydration tanks, scissors used for cutting buds, omega-cut grafting machines, and peat used for callusing. DNA from these samples was extracted and multiplex nested-PCR using primers specific for “Cylindrocarpon” pauciseptatum, Ilyonectria liriodendri and I. macrodidyma-complex (composed of I. alcacerensis, I. estremocensis, I. macrodidyma, I. novozelandica, I. torresensis, and two undescribed species) was used to identify the species present. Ilyonectria liriodendri and I. macrodidyma-complex were detected in hydration tanks, scissors, grafting machines and peat, I. macrodidyma-complex being the most frequent. Additionally, ten grafted cuttings each from five grapevine scion/rootstock combinations were collected from each nursery immediately after callusing, and again after one growing season in a nursery field. Roots of these grafted cuttings and plants were sampled to isolate the fungal pathogens. Only I. torresensis was isolated after callusing, while I. liriodendri, I. novozelandica and I. torresensis were isolated after one growing season, showing the highest incidence at this latter sampling time. Moreover, DNA was extracted from roots and analyzed as described before. Ilyonectria liriodendri and I. macrodidyma-complex were also detected at both sampling times. The use of the multiplex nested-PCR technique improved the detection of I. liriodendri and I. macrodidyma-complex from grafted cuttings and plants in both nurseries. This work shows that the grapevine nursery propagation process should be considered as a potential infection source for black-foot disease pathogens, and confirms that infections caused by Ilyonectria spp. in grapevine planting material increase markedly after one growing season in nursery fields.

Occurrence of Phaeomoniella chlamydospora on grapevine planting material in Sardinia and its control with combined hot water and cyproconazole treatment.

The occurrence of Phaeomoniella chlamydospora was investigated during the propagation process of an Italian nursery, and combined hot water and cyproconazole treatments were carried out to limit its spread in nursery plants. In the three–year period 2005–07, cutting and graft samples (scion cv Sangiovese, rootstock cvs 140Ru in 2005 and 1103P in 2006–07) were taken during the propagation process at several infection risk stages in order to assess the presence of P. chlamydospora. Moreover, in 2005 and 2006 cuttings from esca–symptomatic grapevines (scion cv Sauvignon blanc, rootstock cvs 140Ru in 2005 and 1103P in 2006) were treated at different stages of the propagation process. In 2007, artificially infected 1103P cuttings were treated after inoculation with P. chlamydospora (107 conidia ml-1). Cuttings and callused graftlings were treated by dipping in a hot water bath at 50°C for 30 min (HWT) or in a cyproconazole suspension (0,1 g a.i. l-1) for at least 12 h, in different combinations. At each stage of infection risk and at each treatment woody material was collected and grown in pot, avoiding accidental contamination, or in the field nursery for one season and then destructively examined. DNA was extracted from wood collected at different points along the plant and analysed by nested PCR with Pch-specific primers. One-mm thick woody slices from artificially inoculated cuttings were plated on MEA plus antibiotics and fungicides. Despite the extended wood discoloration, P. chlamydospora occurrence on nursery plants was scarce in the three–year period. Planting material contamination may have resulted from infected mother plants (from 0.0 to 6.7 % of infected cuttings were obtained directly from non-symptomatic mother plants) or from the propagation process, particularly during the stages following grafting (0.0 to 23.3 % of infected grafts). Canes from esca–diseased mother plants were always contaminated, but in quite different percentages (about 30 % of the cuttings examined in 2005, from 1.9 to 4.1 % in 2006 and 2007). However, no final conclusion could be drawn about which stages played a major role in the contamination of nursery plants, due to the low and irregular infection frequencies detected in the propagation process. As regards P. chlamydospora control, HWT performed on cuttings before or after cold storage influenced vegetative growth depending on both the cultivar and the growth conditions, but it was deleterious on callused graftlings. Natural contamination on nursery material in 2005 and 2006 was insufficient to assess the effectiveness of treatments. In 2007, HWT and cyproconzole treatments alone were not effective in reducing the percentage of infection in cuttings that were artificially inoculated. Only cyproconazole immediately followed by HWT significantly reduced the number of infected cuttings, but it was not sufficient to eradicate the pathogen.

Effect of hot-water treatments above 50 °C on grapevine viability and survival of Petri disease pathogens

Rootstocks (41 B Mgt., 140 Ruggeri, 161-49 Couderc, 1103 Paulsen and 110 Richter) and scion/rootstock combinations (Bobal/1103 P, Merlot/110 R, Tempranillo/110 R and Tempranillo/161-49 C) were hot-water treated (HWTed) at 50, 51, 52, 53, or 54 °C for three periods: 30, 45 or 60 min. Four groups of 10 cuttings were treated for each temperature and time, and four additional groups of 10 untreated cuttings were prepared as controls. At the end of the growing season, cutting sprouting and shoot weight were evaluated. In a second experiment, healthy cuttings of ‘110 R’ rootstock were vacuum-inoculated with conidial suspensions (10 6 conidia mL −1) of one isolate of either Phaeomoniella chlamydospora or Phaeoacremonium aleophilum. These cuttings were subjected to the treatments indicated above. Four groups of 10 cuttings were treated for each temperature, time and isolate, with their respective controls. Isolations were made immediately after the treatments and at the end of the growing season, when cutting sprouting and shoot weight were evaluated. Results demonstrated that it is possible to hot-water treat grapevine planting material in Spanish nurseries using protocols with temperatures of up to 50 °C. HWTs at 53 °C are able to eliminate Pa. chlamydospora completely and strongly reduce the re-isolation of Pm. aleophilum from grapevine wood. These findings will contribute to the development of an effective control for Petri disease in grapevine propagating material in Spanish grapevine nurseries.

Grapevine flavescence dorée phytoplasma

Grapevine flavescence dorée phytoplasma

Hot water treatment, trunk diseases and other critical factors in the production of high-quality grapevine planting material

Summary. This review describes the critical factors on which successful grapevine propagation depends and discusses the steps that can be taken to improve the quality of planting material available to growers. Spasmodic occurrences of young vine decline and the failure of planting material have plagued the wine industry since the 1990s. The syndrome now described as Petri disease has been identified as the probable cause of many of the failures, but hot water treatment (HWT) of dormant cuttings (50°C/30 min), for the control of Phaeomoniella chlamydospora and other endogenous pathogens, has also been implicated in the losses. HWT is known to cause a temporary switch to fermentative respiration and early retarded growth in treated material, particularly in Pinot Noir, but the effects of HWT on dormant vine tissue are not yet fully understood. Poor nursery hygiene and poor storage and handling practices during propagation and planting have also been implicated in vine failure. Demand for planting material has exceeded supply and there has been little incentive for nurseries to improve their standards. The quality of planting material could be significantly improved by changing nursery practices, particularly by discontinuing the practice of soaking cuttings in water, treated or untreated, and by improving general standards of nursery hygiene and the management of cool rooms. There is a need to develop a set of universal quality standards for cuttings and rooted vines. Growers also need to be made aware of the characteristics and benefits of high quality planting material.

Survival of Phomopsis viticola in grapevine cuttings after hot water treatment

Hot water treatment (HWT) at 50°C for 30 min reduced the survival of Phomopsis viticola in infected grapevine cuttings from 75.6% to 0.7%. Submerging cuttings in chlorinated water did not affect survival of the pathogen. Survival of P. viticola in cuttings of Sultana and Waltham Cross (cultivars of Vitis vinifera) after hot water treatment varied, indicating that the effects of HWT may be cultivar dependent. The incidence of P. viticola in untreated cuttings of different cultivars and from different sources was also variable. The results indicated that HWT of cuttings can be used to substantially reduce, but not eliminate, infections of P. viticola in grapevine planting material.

Schemes and methods for production and certification of virus‐free grapevine planting material 1

Based on experience in Romania and data from the literature, a scheme for the production of certified grapevine propagating material is presented. In addition, details of the test methods for determining presence of grapevine viruses are described.