FIELD TREATMENT WITH RESISTANCE INDUCERS FOR THE CONTROL OF GRAPEVINE BOIS NOIR

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1 Journal of Plant Pathology (2009), 91 (3), Edizioni ETS Pisa, FIELD TREATMENT WITH RESISTANCE INDUCERS FOR THE CONTROL OF GRAPEVINE BOIS NOIR G. Romanazzi 1, D. D Ascenzo 2 and S. Murolo 1 1 Dipartimento di Scienze Ambientali e delle Produzioni Vegetali, Sezione di Protezione delle Piante, Università Politecnica delle Marche, Via Brecce Bianche, Ancona, Italy 2 ARSSA Servizio Fitosanitario Regionale, Regione Abruzzo, Via Nazionale 38, Villanova, Italy SUMMARY Phytoplasmoses are severe diseases of grapevine and other crops, for which no effective means of control are available. However, plants infected by phytoplasma can undergo spontaneous symptom remission (recovery), a long-known phenomenon. A strategy to reduce the number of symptomatic plants could thus arise from the stimulation of plant defence systems to induce recovery. The aim of the present study was to evaluate the effectiveness of field treatments with resistance inducers to promote recovery in Bois noir (BN) infected grapevines. Five commercial products (Chito Plant, Aliette, Kendal, Bion, and Olivis) described as plant defence promoters were sprayed on the canopy of BN-infected grapevines of cv. Chardonnay. Treatments consisted of weekly sprays in spring-summer 2007 (seven applications) and 2008 (thirteen applications). All treatments increased the number of recovered plants with respect to the control. The best results were obtained with Kendal, Olivis and Bion, while Aliette and Chito Plant performed better in the first than in the second year. Molecular analysis of leaf vein extracts from recovered plants failed to detect the phytoplasma. An induction of host defence is likely to be responsible for the effectiveness of these resistance promoters in the control of BN. Key words: induced resistance, recovery, Vitis vinifera, Bois noir, phytoplasma. INTRODUCTION Phytoplasma are widespread prokaryotes responsible for severe diseases of many plants, generally known as yellows. In grapevine (Vitis vinifera), Flavescence dorée (FD) and Bois noir (BN) are the most common yellows in Europe (Martelli and Boudon-Padieu, 2006). FD, a A2 quarantine disease in the European Plant Protection Corresponding author: G. Romanazzi Fax: g.romanazzi@univpm.it Organization (EPPO) list, occurs in several regions of central and northern Italy, while BN, which is caused by a phytoplasma belonging to the 16SrXII-A subgroup (Candidatus Phytoplasma solani) (Firrao et al., 2005), is widespread in the main Italian viticultural areas (Barba et al., 2006), including Marche and Abruzzi (central Italy) (Romanazzi et al., 2007a, 2007b). To date, there are no known effective means to reduce the incidence of phytoplasma diseases, and even the preventive measures (sanitary selection and use of clean propagating material) associated with treatments against the leafhopper vectors do not reduce BN incidence (Borgo and Angelini, 2002). Heat and hot water treatments, alone or coupled with tissue culture, have also been used in the attempt to eliminate phytoplasma from different hosts (Bianco et al., 2000; Mannini and Gribaudo, 2008). Plants infected by phytoplasma can, however, undergo spontaneous symptom remission, also known as recovery (Caudwell, 1961; Osler et al., 1993). As recovery can be a temporary or permanent symptom remission, recently Maixner (2006b) proposed that a plant can be considered recovered after the minimum of three consecutive years without symptoms. This natural phenomenon, which has been observed in different varieties and viticultural regions (Osler et al., 2003; Garau et al., 2007; Maixner, 2006b; Romanazzi et al., 2007b), appears to be associated with high levels of H 2 O 2 in the leaves of vines recovered from FD (cv. Prosecco), as compared to healthy and FD-infected plants (Musetti et al., 2007). Recovery can also be promoted by exposing grapevines to abiotic stresses, such as transplanting (Osler et al., 1993), partial uprooting or pulling (Romanazzi and Murolo, 2008), and through agronomical practices, such as pruning or pollarding (Borgo and Angelini, 2002; Zorloni et al., 2002). An innovative possibility for the control of grapevine yellows involves spraying the plant canopy with resistance inducers. Induced resistance is a non-specific form of disease resistance in plants that can act against a wide range of pathogens, and can be activated by several non specific inducers, known also as elicitors (Vallad and Goodman, 2004). Some elicitors have systemic activity (induction of generalized resistance) while others only induce local resistance, at the site of application

2 678 Resistance inducers to control Bois noir Journal of Plant Pathology (2009), 91 (3), (Palukaitis and Carr, 2008). There are now a variety of commercial resistance inducers available, some of which are registered in Italy for use on grapevine as plant protectant products. The aim of the present study was to assess the impact of field treatments with resistance inducers on the recovery of BN-infected grapevines. MATERIALS AND METHODS In the framework of a monitoring for grapevine yellows carried out in collaboration with the Phytosanitary Service of Abruzzo (central Italy), a vineyard of cv. Chardonnay near Atri (444 m of altitude) with a BN incidence of ca. 30% in 2006, was selected for field trials. The vineyard was planted in 1990 on a surface of ca. 4,000 m 2 and consisted of 744 vines trained as arbor and spaced 2.5 x 2.5 m. Rainfall is around 500 mm/year mostly in winter (Fig. 1). The vineyard is subjected to ordinary treatments for the control of fungal diseases, and none of the active ingredients applied in these trials had ever been used before. When the vineyard was visually inspected in September 2006, all the vines with symptoms of phytoplasma infection were recorded on a bidimensional map. Ten percent of symptomatic vines (20 samples), representative of the sanitary status of the field, and 12 samples of symptomatic weeds present in the vineyard (5 Convolvulus arvensis, 5 Tussilago farfara, and 2 Cirsium spp.) were collected and analysed molecularly for the presence of phytoplasmas. In September 2007 and 2008, all recovered vines were also analysed. Total DNA was extracted from the veins of symptomatic leaves using the DNeasy Plant Mini Kit (Qiagen, Germany) and amplified with the P1/P7 universal primers (Schneider et al., 1995). A second amplification round followed with the group-specific primers R16(I)F1/R1, R16(III)F2/R1 and R16(V)F1/R1 (Lee et al., 1994), for the detection of stolbur and other grapevine phytoplasma belonging to the 16SrI, 16SrIII and 16SrV groups. The samples that yielded amplicons with R16(I)F1/R1 were used for further characterization, i.e. amplification of the tuf gene and digestion with HpaII at 37 C for 4 h, to determine the tuf type (Schneider et al., 1997). The five commercial products tested in field trials consisted of chitosan (Chito Plant, ChiPro GmbH, Germany), Phosetyl-Al (Aliette, Bayer Crop Science, Germany), two different mixtures of glutathione and oligosaccharines (Kendal, Valagro, Italy and Olivis, Agrisystem, Italy, respectively), and benzothiadiazole (Bion, Syngenta Crop Protection, Switzerland). The chemicals were dissolved in deionized water at their highest advised rates, according to manufacturer s instructions, and were sprayed on the canopy of BN-infected grapevines (Table 1). Plants showing BN symptoms in 2006 were divided into 3 blocks. In 2007 treatments were made weekly from the beginning of June to the middle of July (seven applications) and, in 2008, from the beginning of May to the beginning of August (thirteen applications), by spraying a volume equivalent to 1,000 l/ha using a motorized backpack sprayer (Honda GX 25, Japan). As mentioned, the experiment consisted of 3 completely randomized blocks, with twelve replicates (single plants each) in 2007, and five in In 2008, the inducers were applied to the vines that were treated (or were used as controls) in 2007 and showed disease symptoms in August of the same year. A number of symptomatic plants equal to those that had been sprayed with each product were used as untreated controls to assess the rate of natural recovery. Disease symptoms were recorded on leaves and clusters at the end of July and August 2007 and The data, expressed as percentage of recovered plants, were submitted to an analysis of variance (ANOVA 2) and the means were compared by Duncan s Multiple Range Tests with the MSTAT-C statistical package. The climatic data of the area (minimum, maximum and mean monthly temperatures, and mm rainfall) were kindly provided by Mrs. D. Giuliani and A. Zinni, weather stations of ARSSA Car Scerni (CH), Abruzzo Region. RESULTS In 2006, in the vineyard under investigation, 214 of 744 plants showed typical symptoms of grapevine yellows. The molecular analyses showed that all samples collected were infected by stolbur tuf type II, which was also detected in symptomatic C. arvensis and T. farfara (data not shown). In 2007, all treatments increased the incidence of recovered plants, with respect to the control. During this year, which was characterized by sporadic rainfall from the end of winter till harvest, and temperatures above the seasonal means (Fig. 1), Bion, Olivis, Kendal and Aliette yielded the highest percentages of recovered plants, which were significantly (P 0.05) higher than the control (Table 2). A tendency towards a reduction of symptomatic plants was also shown by Chito Plant. Considering the percentage of naturally recovered plants, which in 2007 was 37.5%, all treatments augmented the number of recovered vines, although not always significantly, with increases ranging from 29.6% (Chito Plant) to 112.0% (Bion). In 2008, the treatments that significantly (P 0.05) increased the recovery with respect to control were Kendal, Bion and Olivis (Table 2). Natural recovery in control vines was 23.1%, whereas chemically-induced recovery ranged from 23.8% (Aliette) to 147.2% (Kendal). Overall, an impressive decrease of sympto-

3 Journal of Plant Pathology (2009), 91 (3), Romanazzi et al. 679 matic plants occurred in the experimental field after the two years of treatments. In particular, in 2008, only 71 out 214 (33%) symptomatic plants occurring at the beginning of the investigations showed grapevine yellows, 12 of which were controls. None of these treatments showed phytotoxic effects. In the experimental plot, in 2008 about 20 newly infected vines (2.7%) were identified. In both years, PCR testing of recovered plants failed to detect the phytoplasma. DISCUSSION Grapevine yellows are diseases that can have important economic impacts on grapevine production (Maixner, 2006a; Martelli and Boudon-Padieu, 2006). BN is endemic in the main viticultural areas of Europe, and it can dramatically decrease the qualitative and quantitative yields of susceptible grapevine cultivars, such as cv. Chardonnay (Borgo and Angelini, 2002). To date, there are no treatments that can sanitize phytoplasma-infected vines. The use of antibiotics as compounds to which phytoplasma can be sensitive has been suggested, but their use in the European Union has been banned. Moreover, the impossibility of cultivating phytoplasma in vitro has increased the difficulties of finding active compounds that can inhibit their growth. Under these circumstances, apart from pruning the symptomatic parts of the plants, one of the few choices is to promote plant resistance. This strategy has been successfully applied to in vitro-grown periwinkle shoots infected with different Candidatus Phytoplasma species, when treated with indole-3-acetic acid (IAA) and indole-3-butyric acid (IBA) (Curkovic Perica, 2008). Both of these auxins induced the recovery of phytoplasma infected periwinkle shoots, and IBA proved to be more effective than IAA. The present study was designed to evaluate the ef- Table 1. Properties of the commercial resistance inducers used in the trials. Commercial product Active ingredient Preharvest interval (days) Suggested rate (kg/ha or l/ha) Advised application interval (days) Aliette Phosetyl-Al yes Registered for use on grapevine in Italy Kendal Glutathione + oligosaccharines yes Chito Plant Chitosan n.a.* yes Bion Acibenzolar-S-methyl (benzothiadiazole) Olivis Glutathione + oligosaccharines *not available no yes Table 2. Effect of field treatment with resistance inducers on the recovery rate of BN-affected grapevines. Trials 2007 Trials 2008 Commercial product Recovered plants (%) Recovery increase viz control (%) Recovered plants (%) Recovery increase viz control (%) Aliette 65.7 ab* bc 23.8 Kendal 71.4 a a Chito Plant 48.6 bc bc 35.1 Bion 79.5 a ab Olivis 74.5 a ab Control 37.5 c c - * Values followed by the same letter are not statistically different, according to Duncan s multiple range test at P 0.05.

4 680 Resistance inducers to control Bois noir Journal of Plant Pathology (2009), 91 (3), Temperature ( C) Rain (mm) Fig. 1. Climate data recorded in 2007 in the area of the study vineyard and historical mean values of last 40 years in the province of Teramo (Abruzzo, central Italy). fects on BN-affected grapevines of treatments with several elicitors. Several trials have been carried out in vineyards to test the effectiveness of resistance inducers in the control of this disease. In Emilia Romagna (northern Italy) Mazio et al. (2008) tested the effectiveness of treatments with several organic fertilizers, based on nitrogen, humic and fulvic acids, and algal extracts applied to the canopy and soil at four different phenological stages, for the control of grapevine yellows on cvs Lambrusco Salamino and Ancellotta. The percentage of recovered plants was affected both by the year and the cultivar, and no significant differences were found between vines treated with these bioactivators and the control. In Sardinia (Italy), Kendal was applied three times in 2005 and 2006 on the canopy of BN-affected cvs Chardonnay and Vermentino vines (Garau et al., 2008). In these trials, there were no significant effects of these treatments on the qualitative and quantitative parameters of healthy, symptomatic and symptomless plants, or on the frequency of the recovered vines. In the present study, we tested a more intense treatment schedule (7-13 applications a year), which proved indeed to influence the number of recovered cv. Chardonnay plants. The best results after two years of application were obtained with the use of Kendal, Bion and Olivis, which decreased the number of symptomatic plants from 35 to 6, 7 and 8 respectively, over the two years of treatment. The application of Aliette and Chito Plant also showed the tendency to increase recovery rates as compared to the control. Several studies have shown that Bion can successfully induce resistance to various pathogens, by increasing the content of pathogenesis-related (PR) proteins in many plant species (Vallad and Goodman, 2004). Bion has also been shown to provide some protection and a delay in symptom appearance in chrysanthemums infected by Ca. P. asteris (D Amelio et al., 2007), a protectant activity against X- disease phytoplasma, and reduced leafhopper survival when applied to Arabidopsis thaliana (Bressan and Purcell, 2005). Both Kendal and Olivis are based on a mixture of glutathione and oligosaccharines. In particular, functions proposed for glutathione in higher plants include storage and transport of reduced sulfur, a protein reductant, a protective role in cellular metabolism, and removal of free radicals. In Phaseolus vulgaris, treatment with exogenous glutathione causes a massive and selective induction of transcription of defence genes encoding enzymes of phytoalexin and lignin biosynthesis, as well as the stimulation of genes encoding cell wall components (Wingate et al., 1988). Chito Plant is based on chitosan, a natural biopolymer with antimicrobial properties and an elicitor of plant resistance against fungi, bacteria and viruses (Kulikov et al., 2006; Romanazzi et al., 2009b). In grapevines, chitosan was shown to induce the production of phenylalanine ammonia lyase and to prime catechin and trans-resveratrol content (Romanazzi et al., 2006). In our trial, Chito Plant showed the tendency to reduce the number of symptomatic plants, compared to the control. Similar results were reported by D Amelio et al. (2007), who observed a delay in symptom development on chrysanthemums infected by Ca. P. asteris after chitosan application. Treatments with resistance inducers carried out on phytoplasma-infected Catharanthus roseus showed different responses of the plants sprayed with Phosetyl-Al, contained in Aliette, and chitosan (Chiesa et al., 2007), the first being more effective. Although the extant pathosystem is different, in our trials chitosan and Phosetyl-Al were not significantly different from each other in both years monitored. Recovery appears to be induced by different factors (Kunze, 1976), among which there are differential varietal propensities (Bellomo et al., 2007; Garau et al., 2007; Romanazzi et al., 2007b), rootstock combinations

5 Journal of Plant Pathology (2009), 91 (3), Romanazzi et al. 681 (Romanazzi and Murolo, 2008), and climatic conditions. In particular, 2007 was one of the driest and hottest periods when compared to the historical climatic data recorded in the Teramo Province (site of the trial), with about a 50% reduction in the rainfall with respect to the mean values from November 2006 to August 2007, and an almost complete lack of rain between June and September The relatively high incidence of recovery in 2007 (37.5%) can be ascribed to stressful climatic conditions that characterized the area during the summer period. Similarly, a significant correlation between recovery rates and high temperatures has been reported, along with an inverse correlation between recovery rate and rainfall from May to August for two cv. Chardonnay vineyards affected by BN in two different Tuscany provinces (Pistoia and Siena) in a four-year investigation (Braccini and Nasca, 2008). The year 2008 was characterized by a cooler summer (data not shown), thus providing conditions that favoured natural recovery at a rate of about 23%. In the recovered plants, molecular testing of leaf veins failed to disclose the presence of phytoplasmas, in agreement with what reported from several Italian and German areas for BN- and FD-infected plants (Osler et al., 2003; Maixner, 2006b; Morone et al., 2007; Romanazzi and Murolo, 2008). Thus, we now confirm the absence of phytoplasmas in leaf veins of recovered symptomless plants. The detection of new symptomatic plants in the course of the trials suggests an active spreading of the disease, fostered by the abundant populations of the BN vector, Hyalesthes obsoletus in the vineyards of the province (Mori et al., 2008) and confirmed by the presence of infected herbaceous hosts (Romanazzi et al., 2009a). So, we cannot exclude that re-infection took place in some of the treated plants, as we have no information on the possible presence in the sprayed plants of compounds that would affect the vector. Tomatoes treated with Bion had a reduction of fungal and bacterial infections and of leaf miners, whose adults preferred untreated plants for ovipositioning (Inbar et al., 1998). In plants treated with Bion, an increase in PR proteins was detected, including peroxidase, lysozymes, chitinase and β-1,3 glucanase. The data obtained in the two-year experiments in the field have allowed us to verify the activity of the tested plant defence inducers. Questions, however, are pending on the toxicity, ecotoxicity and compatibility of these products and the treatment schedule with IPM programs. To answer these concerns, it will be important to plan new studies taking into account different treatment strategies (leaf or soil), the effectiveness of a lower number of applications for some of the products used, and testing further resistance inducers. From a practical point of view, however, it is worth noting that Bion is not registered for use on grapevine, Aliette has a 40 day preharvest interval (and Chardonnay is harvested in central eastern Italy around the middle of August, while the other cultivars are usually in September-October) and that Kendal, Olivis and Chito Plant are registered as promoters of plant resistance and not as plant protection products. Moreover, the mechanisms of action of these elicitors still remain to be investigated. ACKNOWLEDGEMENTS This work was carried out within the projects Varenne funded by the Fondazione Cariverona (Bando 2007), and MIUR PRIN _002. 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