First report of Neofusiccoccum vitifusiforme and presence of other Botryosphaeriaceae species associated with Botryosphaeria dieback of grapevine in Sicily (Italy)

Abstract

Since year 2007, when a grapevine decline, caused by Lasiodiplodia theobromae was reported for the first time in Sicily, vines showing similar declining symptoms have been also found in other grape-growing areas of western and central Sicily. The authors have reported the result of a study on fungi associated with grapevine decline in Sicily, with particular regard to those belonging to the Botryosphaeriaceae. Four species were found to be associated with declining vines, namely Diplodia seriata, Lasiodiplodia sp., Neofusicoccum parvum and Neofusicoccum vitifusiforme, the latter species reported for the first time on Vitis vinifera in Italy.

Headnote

Summary. Since 2007, when a grapevine decline caused by Lasiodiplodia theobromae was reported for the first time in Sicily, vines showing similar declining symptoms have been also found in other grape-growing areas of western and central Sicily. We report the result of a study on fungi associated with grapevine decline in Sicily, with particular regard to those belonging to the Botryosphaeriaceae. Four species were found to be associated with declining vines, namely Diplodia seriata, Lasiodiplodia sp., Neofusicoccum parvum and Neofusicoccum vitifusiforme, the latter species reported for the first time on Vitis vinifera in Italy.

Key words: Diplodia seriata, Lasiodiplodia sp., Neofusicoccum parvum, Vitis vinifera, grapevine trunk disease.

Introduction

Species in the Botryosphaeriaceae Theiss & P. Syd. are cosmopolitan and have been reported as endophytes, parasites, and saprophytes on a broad range of both annual and perennial hosts (Barr, 1972; Punithalingam, 1980; von Arx, 1987; Smith et al., 1996; Burgess et al., 2005; Slippers and Wingfield, 2007) including grapevines (Chamberlain et al., 1964; Lehoczky, 1974, 1988; Hewitt, 1988; Leavitt, 1990, Úrbez-Torres, 2011). Investigations conducted dur- ing the last decade in different countries have shown that actually several species of Botryosphaeriaceae have a pathogenic role in Vitis vinifera causing trunk diseases (Phillips, 2002; van Niekerk et al., 2004, 2006; Taylor et al., 2005; Úrbez-Torres et al., 2008; Úrbez- Torres and Gubler, 2009; Linaldeddu et al., 2010; Pitt et al., 2010), recently reported as Botryosphaeria die- back (Úrbez-Torres, 2011). Geographical distribution of some Botryosphaeriaceae species has been shown to be associated with climate. In particular, Lasiodip- lodia theobromae (Pat.) Griff. & Maubl. is the preva- lent species in warmer grape-growing areas of many countries such as Australia (Taylor et al., 2005; Pitt et al., 2010), California, Arizona, Mexico (Úrbez-Torres et al., 2006, 2008), Egypt (El-Goorani and El Meleigi, 1972), Spain (Aroca et al., 2008) and also in Italy (Bur- ruano et al., 2008).

Among the 21 Botryosphaeriaceae reported to be associated with decline symptoms on grapevine (Úrbez-Torres, 2011), only five species have been to date recognized in Italy on V. vinifera: Botryosphaeria dothidea in Apulia (Carlucci et al., 2009), Marche (Ro- manazzi et al., 2009) and Central Italy (Spagnolo et al., 2011); Diplodia seriata in Molise (Cristinzio, 1978), Apulia (Pollastro et al., 2000) and Central Italy (Spag- nolo et al., 2011); L. theobromae in Sicily (Burruano et al., 2008) and Apulia (Carlucci et al., 2009); Neofu- sicoccum australe in Sardinia (Linaldeddu et al., 2010) and Neofusicoccum parvum in Apulia (Carlucci et al., 2009) and Central Italy (Spagnolo et al., 2011). These species are associated with grapevine decline symp- toms, as late sprout and or dead buds, sub-cortical brown streaking and wedge-shaped wood necrosis, and with "esca" symptoms. After the first report of Botryosphaeria dieback in a vineyard in West Sic- ily by Burruano et al. (2008), similar grapevine de- cline symptoms were also observed in other Sicilian grape-growing areas. Thus, the aim of the present study was to ascertain the occurrence and identity of the Botryosphaeriaceae species associated with Bot- ryosphaeria dieback in Sicily.

Materials and methods

Over the four years (2008--2011) since the first re- port on grapevine decline in Marsala (Trapani), de- clining plants were gradually detected and some of these collected in different grape-growing areas in Sicily: 3 plants cultivar Insolia in Marsala, annually from 2008--2010, and in 2010--2011, 2 plants cv. Mer- lot in Salemi (Trapani), 2 cv. Grillo in Alcamo (Trapa- ni), 2 cv. Alicante Bouchet in Montevago (Agrigento) and 2 cv. Insolia in Milena (Caltanisetta).

Sample collection and fungal isolation

In the summer of each year (July--September), de- clining grapevines were collected and sectioned in several portions both to detect and describe wood symptoms and for isolations. Symptomatic wood portions, after bark removal, were further cut into disks, about 2--3 cm in diameter, which were flame sterilized. Fragments at the margin between the healthy and affected tissue were excised aseptically and plated on 2% malt-extract agar (MEA, Oxoid, Milan, Italy). Petri dishes were kept at 25±1°C in the dark and examined daily for fungal development during two weeks. The isolation frequency (IF) of each fungal genus was calculated using the formula: IF = (Nif/Ntf)x100, where Nif is the number of colo- nies of a given fungus, and Ntf the total number of isolations attempted x100 (Ragazzi et al., 2001).

The Botryosphaeriaceae isolates, which were se- lected on the basis of gross colony morphology, were transferred to PDA (Oxoid) and incubated until fun- gal spore production. Identification of fungal spe- cies was based on the morphology of monosporic colonies and conidial characters. Conidia (100) were observed and measured at 40x magnification with a light microscope equipped with an HRc Axiocam digital camera and accompanying software (Carl Zeiss Ltd, Germany).

The optimum growth temperature of three iso- lates of each morphologically characterized species was determined by incubating the cultures in the dark at temperatures ranging from 5 to 40°C at 5°C intervals, with three replicates per temperature. For each colony two orthogonal diameters were meas- ured after 2 and 3 days, and the colony diameter was expressed as mean radial growth in millimeters (Úrbez-Torres et al., 2008).

Molecular identification

Fungal genomic DNA of all isolates was extracted from monosporic cultures following a standard CTAB- based protocol (O'Donnell et al., 1998). Internal Tran- scribed Spacer (ITS) regions, ITS1 and ITS2, including the 5.8S gene, of the ribosomal DNA (rDNA) operon, were amplified with the primers ITS1-F (Gardes and Bruns, 1993) and ITS4 (White et al., 1990). Part of the translation elongation factor 1-alpha (EF1-?) gene was amplified with the primers EF1-728F and EF1-986R (Carbone and Kohn, 1999). The primers Bt2a and Bt2b (Glass and Donaldson, 1995) were used to amplify a portion of the ?-tubulin (BT) gene. The PCR reactions were performed following the PCR protocol described by Slippers et al. (2004). The ITS-RFLP technique was applied to identify groups among the collected isolates and to select representative isolates for sequencing. The PCR amplicons of the ITS regions were digested separately with CfoI and HaeIII restriction endonucle- ases (Slippers et al., 2007) following the manufacturer's instructions (Fermentas, Milan, Italy). The digestion reaction was incubated at 37°C overnight. The result- ing restriction fragments were separated by electro- phoresis on 2% (w/v) agarose gel and then molecular weights determined. Isolates for which identical RFLP patterns were obtained with both endonucleases were considered to belong to the same RFLP group. ITS, EF1-? and BT regions of a representative isolate of each RFLP group were sequenced in both directions using the same primers as for PCR reactions. Nucleo- tide sequences were compared to GenBank sequences through BLASTn searches.

Pathogenicity

Six-month-old shoots, 8--10 mm in diameter and 30 cm long, were collected from healthy, mature In- solia grapevines, the leaves and tendrils removed, and the shoots surface sterilized with 70% ethanol. Four isolates of each species were used and inoculat- ed onto three shoots. The shoots were first wounded, 10--15 cm from the apex, by removing the bark with a sterile scalpel. A 6-mm-diam. plug from of a 7-day- old colony on PDA was then placed on each wound and immediately covered with Parafilm. Control shoots were inoculated with non-colonised plugs of PDA. All inoculated shoots, were placed into a 3-cm- diameter tubes containing 200 mL of tap water, and covered with plastic bags to maintain humidity. Af- ter 21 days at 25°C with natural light, each shoot was evaluated for the length of vascular discoloration around the point of inoculation. Data were submit- ted to ANOVA and to the Student's t-test. Re-isola- tions from symptomatic tissues were carried out on PDA to fullfill Koch's postulates.

Results and discussion

Declining grapevines observed in the five vine- yards of West and Central Sicily showed in field late sprouting and/or mortality bud, delayed growth, cankers and dieback. In particular, bud and canopy symptoms were more evident in spring, since as the season progressed the grapevines seemed to recover their vegetative growth. Longitudinal sections of symptomatic samples always showed, along the whole length or nearly so of the trunk, brown wood necrosis (Figure 1a) that was often wedge-shaped in cross section (Figure 1b). In addition, sub-cortical longitudinal brownish discolored bands were of- ten detected (Figure 1c). Only in Milena and Salemi samples, white rot starting from pruning wounds spreading along the trunk was also observed.

From the symptomatic wood of all sampled grape- vines, a total of 1079 fungal colonies were isolated (Table 1). Among these, Botryosphaeriaceae species predominated (670 isolates) with IF ranging between 23.5% (Alcamo samples) and 76.5% (Milena samples). Saprophytic and wood contaminant fungi belonging to Penicillium, Aspergillus and Alternaria were also present, while colonies of Acremonium, Cladosporium, Fusarium, Phoma, Rhizopus and Fomitiporia mediterra- nea were sporadically observed. Contrary to what is often reported by other authors (Pollastro et al., 2000; van Niekerk et al., 2006; Úrbez-Torres et al., 2006; Pitt et al., 2010), other causal agents of grapevine decline such as Phaeomoniella chlamydospora, Phaeoacremonium spp. and Eutypa lata were never isolated.

On the basis of colony and conidial morphol- ogy, and optimum growth temperature, Botryospha- eriaceae isolates were classified into four groups (Ta- ble 2), which were supported by the ITS-RFLP profiles (Figure 2). BLASTn searches of the ITS sequences of the four selected isolates showed a high homology with D. seriata (99%), L. theobromae (98%), N. parvum (99%) and Neofusicoccum vitifusiforme (99%) (Table 3). Sequences from EF1-? and ?-tubulin gene regions were used to confirm identification that could not be clearly resolved with ITS sequence. The comparison of sequence data of both EF1-? and ?-tubulin with those in GenBank confirmed the identification based on ITS sequences, with the exception of L. theobromae. Since a phylogenetic analysis is in progress in order to distinguish the two potential cryptic species in L. theobromae (Alves et al., 2008), we report this species as Lasiodiplodia sp.

Among these four species, D. seriata was associat- ed with all samples from various grapevine growing areas (Table 1). In particular, it was the only species isolated from the sub-cortical longitudinal brown bands and, exclusively in those of Alcamo (23.5%) and Milena (76.5%), also from the wood necrosis. This species was also obtained from wood necrosis of grapevines in Marsala, Montevago and Salemi, but not singularly and in different percentages (5.6, 2.5 and 44.5, respectively). Lasiodiplodia sp., on the contrary, was obtained both from Marsala and Sale- mi samples, with IF of 43.8% and 18.9%, respectively. N. parvum was only and sporadically isolated from Marsala grapevines (IF 1.6%), while N. vitifusiforme, from Montevago samples, with a high isolation per- centage (57%).

With regard to the preliminary tests of patho- genicity, the inoculated fungi caused vascular dis- coloration, extending differently both upward and downward from the inoculation point, in the sam- pled shoots (Figure 3). However, the length of vas- cular discolorations was very variable both between the different species and within each species. The control shoots developed only a slight discoloura- tion only around the wound site. ANOVA analysis showed significant differences in the extent of vas- cular discoloration between control and inoculated shoots, but no significant differences between Bot- ryosphaeriaceae species (Table 4). The inoculated fungi were always reisolated from the inoculated canes and no botryosphaeriaceous fungi were reiso- lated from the control. These assays, even if with low number of replicates, allowed to ascertain the patho- genicity of the assayed Botryosphaeriaceae species on V. vinifera.

Our results show several Botryosphaeriaceae species associated with Botryosphaeria dieback in Sicily, as well as already observed in other countries (Úrbez-Torres et al., 2006; Candolfi-Arballo, et al., 2010; Pitt et al., 2010; Úrbez-Torres, 2011); this could be due to environmental conditions of every grape- growing area.

Regarding N. vitifusiforme, reported in V. vinif- era for first the time in South Africa (van Niekerk et al., 2004), is now frequently associated with Botry- osphaeria dieback in several grape-growing areas worldwide: Spain (Luque et al., 2009), New Mexico (Candolfi-Arballo, et al., 2010) and USA (Úrbez-Tor- res, 2011). The occurrence of N. vitifusiforme on Olea europaea in South Italy (Lazzizzera et al., 2008) shows the capability of this species to colonize different hosts, and may confirm the hypothesis of the role of secondary host as a inoculum source for grapevine trunk disease pathogens (Cloete et al., 2011). At pre- sent, N. vitifusiforme has been also recently reported as pathogen in fruit trees: Prunus spp. (Damm et al., 2007), Malus and Pyrus spp. (Cloete et al., 2011), O. europaea (Lazzizzera et al., 2008; Úrbez-Torres et al., 2012) and in blueberry (Vaccinium corymbosum, Kong et al., 2010), mostly associated with host wood necro- sis. To date, this is the first report of N. vitifusiforme in V. vinifera in Italy.

Acknowledgments

We want to thank Benedetto Linaldeddu for sup- porting our molecular tests.

References

Literature cited

Alves A., P.W Crous., A. Correia, A.J.L. Phillips, 2008. Mor- phological and molecular data reveal cryptic speciation in Lasiodiplodia theobromae. Fungal Diversity 2, 1--13.

Aroca A., R. Raposo, D. Gramaje, J. Armengol, S. Martos and J. Luque, 2008. First report of Lasiodiplodia theobromae associ- ated with decline of grapevine rootstock mother plants in Spain. Plant Disease 92, 832.

von Arx J.A. 1974. The Genera of Fungi Sporulating in Pure Col- ture. 2th edition, J. Cramer, Vaduz, Lichtenstein, 315 pp

Barr M.E., 1972. Preliminary studies on the Dothideales in tem- perate North America. Contributions to the University of Michigan Herbarium 9, 523?638.

Burgess T.I., P.A. Barber and G.E.St.J. Hardy, 2005. Botryospha- eria spp. associated with eucalypts in Western Australia including description of Neofusicoccum macroclavatum sp. nov. Australasian Plant Pathology 34, 557?567.

Burruano S., V. Mondello, G. Conigliaro, A. Alfonzo, A. Spag- nolo and L. Mugnai, 2008. Grapevine decline in Italy caused by Lasiodiplodia theobromae. Phytopathologia Mediter- ranea 47, 132?136.

Candolfi-Arballo O., C.Valenzuela-Solano, W.D. Gubler and R. Hernández-Martínez, 2010. Botryosphaeriaceae species associated with grapevine decline in Mexico. Phytopatho- logia Mediterranea 49, 105?106.

Carbone I. and L.M. Kohn, 1999. A method for designing primer sets for speciation studies in filamentous ascomy- cetes. Mycologia 91, 553--556.

Carlucci A., F. Lops, M.L. Raimondo, V. Gentile, M. Mucci and S. Frisullo, 2009. The Botryosphaeria species from vineyards of Apulia. Phytopathologia Mediterranea 48, 180.

Chamberlain, G.C., R.S. Willison, J.L. Towsend, and J.H. De Ronde, 1964. Two fungi associated with the dead arm dis- ease of grapes. Canadian Journal of Botany 42, 351?355.

Cloete M., P.H. Fourie, U. Damm, P.W. Crous and L. Mostert, 2011. Fungi associated with die-back symptoms of apple and pear trees, a possible inoculum source of grapevine trunk disease pathogens. Phytopathologia Mediterranea 50, S176-S190.

Cristinzio G., 1978. Gravi attacchi di Botryosphaeria obtusa su vite in provincia di Isernia. Informatore Fitopatologico 6, 21--23.

Damm U., P.W. Crous, P.H. Fourie, 2007. Botryosphaeriaceae as potential pathogens of prunus species in South Africa, with descriptions of Diplodia africana and Lasiodiplodia plu- rivora sp. nov. Mycologia 99, 664?680.

El-Goorani M.A. and M.A. El Meleigi, 1972. Dieback of grape- vine by Botryodiplodia theobromae Pat. in Egypt. Phytopatho- logia Mediterranea 11, 210?211.

Gardes M. and T.D. Bruns, 1993. ITS primers with enhanced specificity for Basidiomycetes: application to the identifica- tion of mycorrhizae and rust. Molecular Ecology 2, 113?118.

Glass N.L. and G.C. Donaldson, 1995. Development of primer sets designed for use with the PCR to amplify conserved genes from filamentous Ascomycetes. Applied and Environ- mental Microbiology 61, 1323--1330.

Hewitt W.B., 1988. Diplodia cane and bunch rot. In: Compen- dium of Grape Diseases (R.C. Pearson, A.C. Goheen, ed.), American Phytopathological Society Press, St. Paul, MN, USA, 25?26.

Kong C.S., X.L. Qiu, K.S. Yi and X.F. Yu, 2010. First Report of Neofusicoccum vitifusiforme causing Blueberry blight of blueberry in China. Plant Disease 94, 13732--13732.

Lazzizzera C., S. Frisullo, A. Alves and A.J.L. Phillips, 2008. Morphology, phylogeny and pathogenicity of Botryospha- eria and Neofusicoccum species associated with drupe rot of olives in southern Italy. Plant Pathology 57, 948?956.

Leavitt G.M., 1990. The occurrence, distribution, effects and control of Botryodiplodia theobromae on Vitis vinifera in Cali- fornia, Arizona and northern Mexico. PhD Thesis, Univer- sity of California, Riverside, CA, USA.

Lehoczky L., 1974. Black dead-arm disease of the grapevine caused by Botryosphaeria stevensii infection. Acta Phyto- pathologica Academiae Scientiarum Hungaricae 9, 319?327.

Lehoczky L., 1988. Black dead arm. In: Compendium of Grape Diseases. (Pearson R.C., Goheen A.C., ed.). APS Press, St. Paul, MN, USA, 35.

Linaldeddu B.T, B. Scanu, A. Schiaffini and S. Serra, 2010. First report of Neofusicoccum australe associated with grapevine cordon dieback in Italy. Phytopathologia Mediterranea 49, 417?420.

Luque J., S. Martos, A. Aroca, R. Raposo and F. Garcia-Figueres, 2009. Symptoms and fungi associated with declining ma- ture grapevine plants in northeast Spain. Journal of Plant Pathology 91, 381--390.

O'Donnell K., E. Cigelnik and H.I. Nirenberg, 1998. Molecular systematics and phylogeography of the Gibberella fujikuroi species complex. Mycologia 90, 465?493.

Phillips A.J.L., 2002. Botryosphaeria species associated with diseases of grapevines in Portugal. Phytopathologia Medi- terranea 41, 3?18.

Pitt W.M., R. Huang, C.C. Steel and S. Savocchia, 2010. Identi- fication, distribution and current taxonomy of Botryospha- eriaceae species associated with grapevine decline in New South Wales and South Australia. Australian Journal of Grape and Wine Research 16, 258?271.

Pollastro S., C. Dongiovanni, A. Abbatecola and F. Fare- tra, 2000. Observations on the fungi associated with esca and on spatial distribution of esca-symptomatic plantsin Apulian (Italy) vineyards. Phytopathologia Mediterranea 39, 206?210.

Punithalingam E., 1980. Plant diseases attributed to Botryodip- lodia theobromae. In: Bibliotheca Mycologica. J. Cramer, Ber- lin, Germany, 112 pp.

Ragazzi A., S. Moricca, P. Capretti, I. Dellavalle, F. Mancini and E. Turco, 2001. Endophytic fungi in Quercus cerris: isolation frequency in relation to phenological phase, tree health and the organ affected. Phytopathologia Mediterranea 40, 165?171.

Romanazzi G., S. Murolo, L. Pizzichini and S. Nardi, 2009. Esca in young and mature vineyards, and molecular di- agnosis of the associated fungi. European Journal of Plant Pathology 125, 277--290.

Slippers B. and M.J. Wingfield, 2007. Botryosphaeriaceae as en- dophytes and latent pathogens of woody plants: diversity, ecology and impact. Fungal Biology Reviews 21, 90?106.

Slippers B., P.W. Crous, S. Denman, T.A. Coutinho, B.D. Wing- field and M.J. Wingfield, 2004. Combined multiple gene genealogies and phenotypic characters differentiate several species previously identified as Botryosphaeria dothidea. My- cologia 96, 83?101.

Slippers B., W.A. Smit, P.W. Crous, T.A. Coutinho, B.D. Wing- field and M.J. Wingfield, 2007. Taxonomy, phylogeny and identification of Botryosphaeriaceae associated with pome and stone fruit trees in South Africa and other regions of the world. Plant Pathology 56, 128?139.

Smith H., M.J. Wingfield and O. Petrini, 1996. Botryosphaeria dothidea endophytic in Eucalyptus grandis and Eucalyptus nitens in South Africa. Forest Ecology and Management 89, 189?195.

Spagnolo A., G. Marchi, F. Peduto, A.J.L. Phillips and G. Surico, 2011. Detection of Botryosphaeriaceae species within grape- vine woody tissues by nested PCR, with particular empha- sis on the Neofusicoccum parvum/N. ribis complex. European Journal of Plant Pathology 129, 485--500.

Taylor A., G.E.StJ. Hardy, P. Wood and T. Burgess, 2005. Identi- fication and pathogenicity of Botryosphaeria species associ- ated with grapevine decline in Western Australia. Australa- sian Plant Pathology 34, 187?195.

Úrbez-Torres J.R., 2011. The status of Botryosphaeriaceae spe- cies infecting grapevines. Phytopathologia Mediterranea 50, S5?S45.

Úrbez -Torres J.R. and W.D. Gubler, 2009. Pathogenicity of Bot- ryosphaeriaceae species isolated from grapevine cankers in California. Plant Disease 93, 584?592.

Úrbez-Torres J.R., G.M. Leavitt, T.M. Voegel and W.D. Gubler, 2006. Identification and distribution of Botryosphaeria spp. associated with grapevine cankers in California. Plant Dis- ease 90, 1490?1503.

Úrbez -Torres J.R., G.M. Leavitt, J.C. Guerrero, J. Guevara and W.D. Gubler, 2008. Identification and pathogenicity of La- siodiplodia theobromae and Diplodia seriata, the causal agents of bot canker disease of grapevines in Mexico. Plant Disease 92, 519?529.

Úrbez-Torres J.R., F. Peduto and W.D. Gubler, 2012. Olive trees, a potential alternative host for grapevine trunk disease pathogens. Phytopathologia Mediterranea 51, 432-433.

van Niekerk J.M., P.W. Crous, J.Z. Groenewald, P.H. Fourie and F. Halleen, 2004. DNA phylogeny, morphology and patho- genicity of Botryosphaeria species on grapevines. Mycologia 96, 781?798.

van Niekerk J.M., P.H. Fourie, F. Halleen and P.W. Crous, 2006. Botryosphaeria spp. as grapevine trunk disease pathogens. Phytopathologia Mediterranea 45, S43?S54.

White T.J., T. Bruns, S. Lee and J. Taylor, 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: PCR Protocols: a Guide to Methods and Ap- plications (M.A. Innis, D.H. Gelfand, J.J. Sninsky, T.J. White, ed.), Academic Press, San Diego, CA, USA, 315?322.

Accepted for publication: May 25, 2013