Development of isolate‐specific markers for Neofusicoccum parvum and N. luteum and their use to study rainwater splash dispersal in the vineyard

Unique bands were identified in single isolates of Neofusicoccum parvum and Neofusicoccum luteum using universally primed polymerase chain reaction (UP‐PCR) analysis of isolates obtained from grapevines and non‐grapevine hosts in New Zealand, Australia, South Africa and the USA. Primers were designed to amplify a 1550 bp portion of the 1573 bp marker band from N. parvum isolate B2141 and a 510 bp portion of the 524 bp marker band from N. luteum isolate G51a2. A PCR‐RFLP assay was developed to distinguish the N. parvum isolate B2141 from other N. parvum isolates, based on a polymorphism found in the marker band using the TaqI restriction endonuclease. For N. luteum isolate G51a2, the designed primers were specific at an annealing temperature of 63°C in the PCR. The sensitivity threshold of the N. parvum and N. luteum isolate‐specific markers was 50 pg and 5 pg, respectively, when used in standard PCR with purified genomic DNA. The sensitivity of the N. parvum isolate‐specific marker was increased to 0·5 pg by nested PCR. The specificity test of both isolate‐specific markers with six other Botryosphaeriaceae spp. showed that they were specific to their respective species and isolates. Both markers were able to detect the conidia of N. parvum and N. luteum marker isolates in rainwater samples collected at different distances from an inoculation point in the vineyard. The results showed that rain splash could disperse the conidia of both of these species up to 2 m from the inoculum point in a single rainfall event.     

Characterization of Botryosphaeriaceae species associated with diseased loquat (Eriobotrya japonica) in Spain

Loquat (Eriobotrya japonica) is an important subtropical fruit crop in Spain and other Mediterranean countries. In recent years, characteristic symptoms of branch canker and dieback have been observed in the main cultivated areas of loquat in Spain. The goal of this study was to identify and characterize the species of Botryosphaeriaceae associated with these symptoms. For this, 36 affected orchards were surveyed between 2010 and 2011 in six provinces of southeastern Spain. Eighty‐four isolates belonging to the family Botryosphaeriaceae were recovered from samples with symptoms. These isolates were characterized by means of phenotypical studies, DNA sequence analyses of the internal transcribed spacer (ITS) and part of the translation elongation factor 1‐α regions, and pathogenicity tests. Ten fungal species were identified: Diplodia malorum, Diplodia olivarum, Diplodia seriata, Diplodia pseudoseriata/Diplodia alatafructa, Diplodia sp., Dothiorella sarmentorum, Neofusicoccum mediterraneum, N. parvum, Spencermartinsia plurivora and S. viticola. In addition, Diplodia eriobotryicola and Dothiorella eriobotryae are newly described. The most frequent species isolated from cankers was D. seriata and, as far as is known, this is the first report of D. malorum, and species belonging to the complex D. pseudoseriata/D. alatafructa, in Spain. All species were pathogenic to 1‐year‐old loquat plants cv. Algerie, with Diplodia sp. and S. viticola as the most virulent.     

Botryosphaeriaceae occurring on native Syzygium cordatum in South Africa and their potential threat to Eucalyptus

Eight species of the Botryosphaeriaceae (canker and dieback pathogens) were identified on native Syzygium cordatum in South Africa, based on anamorph morphology, ITS rDNA sequence data and PCR-RFLP analysis. The species identified were Neofusicoccum parvum, N. ribis, N. luteum, N. australe, N. mangiferae , Botryosphaeria dothidea, Lasiodiplodia gonubiensis and L. theobromae . Their pathogenicity on S. cordatum seedlings and a Eucalyptus grandis  ×  camaldulensis clone was determined in glasshouse inoculation trials. Isolates of all identified species, except one of N. mangiferae , were more pathogenic on the Eucalyptus clone than on S. cordatum . Some of the species that cross-infected these hosts, such as N. ribis, N. parvum and L. theobromae , were amongst the most pathogenic on the Eucalyptus clone, while B. dothidea and L. gonubiensis were the least pathogenic. Results of this study illustrate that species of the Botryosphaeriaceae from native hosts could pose a threat to introduced Eucalyptus spp., and vice versa .     

Distribution and characterization of Streptomyces species causing potato common scab in Germany

Field‐grown potatoes showing scab infections were sampled in two successive years and analysed for prevailing Streptomyces strains. In 2008 and 2009, 293 Streptomyces isolates were collected in Germany and analysed for morphology, pathogenicity and strain type. Isolates varied in mycelium colour, sporulation and pigmentation. Based on their morphology, no clear differentiation of species was possible. At the genetic level, sampled isolates, as well as a number of type strains from culture collections, were characterized by PCR using 16S rRNA‐specific primers and PCR‐RFLP of the 16S–23S internal transcribed spacer (ITS) region with Hpy99I. Using this fingerprinting approach, Streptomyces species could be differentiated genotypically. The data from this study show that diversity among scab‐causing species in Germany is much higher than previously thought. Isolates belonged to various Streptomyces spp. previously associated with common scab. This is apparently the first report of pathogenic strains of S. europaeiscabiei, S. stelliscabiei, S. acidiscabiei, S. turgidiscabiei and S. bottropensis within Germany. Streptomyces europaeiscabiei was the predominant species found. Other scab‐causing species were identified, but their local distribution was uneven. For most of the isolates, the presence of the txtAB gene was demonstrated, indicating pathogenicity. This analysis is one of the first reports to examine the distribution of common scab‐causing species in Germany.     

Detection of Botryosphaeriaceae species within grapevine woody tissues by nested PCR, with particular emphasis on the Neofusicoccum parvum/N. ribis complex

Several species of Botryosphaeriaceae and Phaeomoniella chlamydospora are common agents of grapevine decline worldwide. Currently, the use of culture independent PCR based techniques for detection of Botryosphaeriaceae within grapevine tissues has been limited to Botryosphaeria dothidea. In the present study, two Botryosphaeriaceae specific nested PCR assays were developed. One with a narrow target range, to detect Neofusicoccum parvum and the closely related species complex (Neofusicoccum parvum/N. ribis sensu Pavlic et al. Molecular Phylogenetics and Evolution 51:259–268, 2009) and another, with a wider range, to detect all 17 species of Botryosphaeriaceae which have been reported as potential wood pathogens of grapevine. The effectiveness of these assays was validated in vivo on naturally infected wood samples collected from standing vines and dormant grafted rooted cuttings commercialized in Italy by different nurseries in different years. All samples were also screened by means of a previously published nested PCR assay specific for Phaeomoniella chlamydospora. It was found that: 1) propagation material may play an important role as source of primary inoculum, not only of Phaeomoniella chlamydospora, as previously reported, but also for members of the Botryosphaeriaceae, among which Neofusicoccum parvum, Botryosphaeria dothidea and Diplodia seriata are the most common, and 2) multiple infections by different species belonging to Botryosphaeriaceae and/or Phaeomoniella chlamydospora occur frequently both in standing vines and propagation material. This last finding supports the hypothesis that at least some of the non-specific symptoms of grapevine decline may be due to the presence of different pathogens within host tissues.     

Genetic variability and pathogenicity of Rhizoctonia solani associated with black scurf of potato in New Zealand

Isolates (a total of 129) of Rhizoctonia solani were collected from black scurf on potato tubers from different potato‐growing regions in New Zealand. Sequence analysis of the nuclear ribosomal DNA internal transcribed spacer (rDNA–ITS) regions from these isolates identified three anastomosis groups (AGs), AG‐3PT, AG‐2‐1 and AG‐5. Isolates classified as AG‐3PT were widely distributed, whereas AG‐2‐1 and AG‐5 were confined to distinct locations. Sequence heterogeneity was identified in the ITS regions of 100 AG‐3PT and AG‐2‐1 isolates. Variation in the sequence and length of the rDNA–IGS1 region was also observed for selected isolates of AG‐3PT and AG‐2‐1. Phylogenetic studies found all AG‐2‐1 isolates belong to AG‐2Nt, a subset of AG‐2‐1 previously associated with solanaceous crops in other countries. AG‐2‐1 isolates were consistently more aggressive than those of AG‐3PT. Delayed emergence, severe infection on stolons, formation of aerial tubers and considerable yield losses were associated with AG‐2‐1, but they caused negligible black scurf. In contrast, AG‐3PT caused black scurf on progeny tubers but variable effects on stem emergence and stolons. Furthermore, AG‐2‐1 isolates caused severe tuber malformation, but isolates of other AGs did not. This is the first report on the AG composition, genetic variability and pathogenicity of R. solani isolates associated with black scurf of New Zealand potatoes.     

Aetiology of branch dieback,panicle and shoot blight of pistachio associated with fungal trunk pathogens in southern Spain

Pistachio represents an emerging nut crop across the Mediterranean basin. In Spain, pistachio has been traditionally cultivated in marginal-dry areas with unfavourable climatic conditions for plant diseases. Consequently, little attention has been given to research on pistachio diseases until recently. Symptoms of branch dieback and cankers, and shoot and panicle blight have been recently observed in commercial pistachio orchards across southern Spain. In this study, 10 commercial pistachio orchards showing disease symptoms were surveyed between 2017 and 2018. Botryosphaeriaceae fungi were consistently isolated from affected shoots, among other fungal families with minor relevance. Representative isolates of each family were characterized based on colony and conidial morphology, optimum growth temperature, and the comparison of DNA sequence data (ITS, LSU, EF, TUB2, and ACT genomic regions). Detached and attached shoots, and attached panicles of pistachio cv. Kerman were inoculated with mycelial plugs or conidial suspensions to demonstrate the pathogenicity of the selected isolates. Botryosphaeria dothidea, Lasiodiplodia pseudotheobromae, Neofusicoccum mediterraneum, N. parvum, Diaporthe neotheicola, Diaporthe sp., Eutypa lata, Eutypa sp., Cytospora sp., and Phaeoacremonium minimum were identified. P. minimum had the highest optimum growth temperature (29.6 °C) and Cytospora sp. the lowest (21–22 °C). Botryosphaeriaceae isolates showed the largest lesions on inoculated shoots, with L. pseudotheobromae being the most aggressive, followed by Neofusicoccum species. Panicles inoculated with N. mediterraneum showed blight symptoms and canker formation 6 weeks after inoculation, without significant differences in aggressiveness between isolates. This work reports relevant information about this emerging disease in the novel Spanish pistachio-growing areas.     

Botryosphaeriaceae species causing dieback on Annonaceae in Brazil

In Brazil, the Annonaceae species Annona muricata, A. squamosa, A. cherimola and atemoya (a hybrid of A. cherimola and A. squamosa) are cultivated in several regions, and produce fruits that are highly appreciated by consumers and are of great economic importance. Among the several diseases that can affect these crops, dieback is one of the most important, causing damage and, in the most severe cases, death of the plants. Due to the lack of suitable diagnostic studies up to now, this work aimed to identify the Botryosphaeriaceae species that cause dieback on Annonaceae in Brazil. Based on combined phylogenetic analyses of ITS, TEF-1α, TUB2 and RPB2, eight species of Botryosphaeriaceae were identified, namely Lasiodiplodia brasiliense, L. crassispora, L. hormozganensis, L. iraniensis, L. pseudotheobromae, L. subglobosa, L. theobromae and Pseudofusicoccum stromaticum. All species found in this study were pathogenic and caused symptoms of necrosis in stems and dieback. Thus, this study confirms species of Botryosphaeriaceae as causal agents of dieback on Annonaceae in Brazil.     

Drought × disease interaction in Eucalyptus globulus under Neofusicoccum eucalyptorum infection

Eucalyptus species are widely spread over the world, being extensively planted and exploited by industries. Drought and pathogens are known to affect the establishment and productivity of Eucalyptus plantations worldwide. The aim of this work was to evaluate the pathogenicity of Neofusicoccum eucalyptorum in drought‐stressed and well‐watered E. globulus plants. The effect of a previous drought priming step and the role of water status at the time of inoculation were evaluated. Lesion length, plant growth and physiological parameters (relative water content, water potential, photosynthetic pigments and lipid peroxidation) were determined. The results indicate that water‐stressed plants were more susceptible to N. eucalyptorum than non‐stressed ones. However, this response was particularly relevant when the plants were inoculated while water limitation was already occurring. Moreover, drought‐primed plants were slightly more resistant to fungal infection than the non‐primed ones. This study reinforces the importance of exploring drought × disease interaction in Eucalyptus and the underlying physiological responses involved in plant performance.     

Dothistroma needle blight: an emerging epidemic caused by Dothistroma septosporum in Colombia

Plantation forestry in Colombia is based mainly on non‐native species of Pinus and Eucalyptus. Since 2008, a disease with symptoms similar to those of dothistroma needle blight (DNB) has been found affecting large areas planted to Pinus spp. The aim of this study was to identify the causal pathogen as well as to document the levels of disease incidence and severity. Isolates from each of three forestry zones, collected from different host species, were compared based on rDNA sequence of the ITS regions. These were conclusively identified as Dothistroma septosporum, one of two Dothistroma spp. known to cause DNB. Susceptibility was greatest on low elevation Pinus tecunumanii followed by Pinus kesiya and Pinus oocarpa. Pinus maximinoi and high elevation P. tecunumanii showed tolerance to D. septosporum. The disease incidence in the different zones varied significantly with the North zone being the most severely affected. This constitutes the first report of disease distribution and susceptibility of hosts, as well as the first consideration of the relative importance of D. septosporum in Colombia.     

Phoma leaf spot of wasabi (Wasabia japonica) caused by Leptosphaeria biglobosa

A leaf spot disease on wasabi plants grown in commercial greenhouses in the Fraser Valley of British Columbia was characterized. Mycelial growth and pycnidial formation were observed within lesions when leaves were incubated under conditions of high humidity. Isolation from diseased tissues consistently yielded colonies of a Phoma species. Sequence analysis of the rDNA internal transcribed spacer (ITS1‐5.8S‐ITS2) region of eight isolates showed 100% nucleotide sequence identity with Phoma wasabiae and Leptosphaeria biglobosa subspecies ‘occiaustralensis’ and 99.2% identity with L. biglobosa ‘canadensis’. Pathogenicity studies on wasabi leaves showed that wounding greatly facilitated infection and enhanced lesion development for most isolates but was not required for all isolates. Chlorotic areas appeared around the inoculation sites within 4 days, followed by necrosis. Isolates displayed a range of virulence, from weakly to highly virulent, on wasabi leaves. Similar results were observed on leaves of canola cultivar Westar, i.e. wounding significantly increased lesion size and isolates displayed a range of virulence. An isolate of Leptosphaeria maculans ‘brassicae’ from canola was highly virulent on wasabi and canola leaves, causing lesions similar to those of L. biglobosa ‘occiaustralensis’ while an isolate of L. biglobosa ‘canadensis’ from canola was weakly virulent on both hosts and required wounds to infect. These results demonstrate that isolates of L. biglobosa ‘occiaustralensis’ from wasabi are as virulent as L. biglobosa ‘canadensis’ on wasabi and canola leaves but in some cases were comparable in virulence to L. maculans ‘brassicae’.     

Variability in aggressiveness of Quambalaria pitereka isolates

Quambalaria shoot blight, caused by the fungal pathogen Quambalaria pitereka, is a serious disease of eucalypt plantations in Australia. The aggressiveness of four Q. pitereka isolates was compared on a range of host genera, species, provenances and clones. Isolates differed substantially in their aggressiveness, with two consistently showing higher levels of aggressiveness based on incidence and severity of disease and lesion size. Isolates derived from Corymbia citriodora subsp. variegata (Ccv) and C. torelliana were shown to have a relatively restricted host range, with lesions but no sporulation found on Eucalyptus species, Angophora species other than A. costata and Corymbia species other than Ccv, the host of origin. The level of aggressiveness toward the different provenances of spotted gum and C. torelliana varied between isolates and there was evidence of some isolate × host interaction within provenances of Ccv. The two methods of inoculation used in this study, spray and spot inoculation, gave similar results. However, the fact that the spot inoculation method was labour‐intensive was a disadvantage limiting the numbers of isolates and hosts that can be tested.     

Virulence affected by assay parameters during grapevine pathogenicity studies with Botryosphaeriaceae nursery isolates

This study assessed the symptoms that developed when 114 Botryosphaeriaceae isolates from grapevine nursery plant materials were used to inoculate excised green shoots and 1‐year‐old rooted canes of Sauvignon blanc. The experiments showed that all isolates and species were able to produce lesions. Overall, the Neofusicoccum species were shown to be highly pathogenic in both tissue types while the Diplodia species were highly pathogenic on canes but not on green shoots. Isolates of the most prevalent species, N. luteum and N. parvum, showed varying lesion lengths on excised green shoots and canes. An evaluation of the factors associated with lengths of lesions showed that they were significantly affected by experimental batch which reflected inherent host and environmental factors over time. Reisolation from inoculated canes also indicated that most isolates of all species except D. seriata were able to spread internally beyond the lesions. Genetic variability analysis using UP‐PCR showed that N. luteum isolates were genetically diverse but no association was observed between the phylogenetic group and degree of pathogenicity caused by the isolates. This study demonstrated that all Botryosphaeriaceae species from grapevine nurseries were pathogenic to grapevines and that the lesion lengths varied between species, among isolates within a species and among nursery sources, and was affected by the test method.     

A quantitative real‐time PCR assay for detection of Colletotrichum lindemuthianum in navy bean seeds

Bean anthracnose is a seedborne disease of common bean (Phaseolus vulgaris) caused by the fungal pathogen Colletotrichum lindemuthianum. Using seed that did not test positive for the pathogen has been proven to be an effective strategy for bean anthracnose control. To quantify the extent of anthracnose seed infection, a real‐time PCR‐based diagnostic assay was developed for detecting C. lindemuthianum in seeds of the commercial bean class navy bean. The ribosomal DNA (rDNA) region consisting of part of the18S rDNA, 5^.8S rDNA, internal transcribed spacers (ITS) 1, 2 and part of the 28S rDNA of seven races of C. lindemuthianum, 21 isolates of Colletotrichum species and nine other bean pathogens were sequenced with the universal primer set ITS5/ITS4. Based on the aligned sequence matrix, one primer set and a probe were designed for a SYBR Green dye assay and a TaqMan MGB (minor groove binder) assay. The primer set was demonstrated to be specific for C. lindemuthianum and showed a high sensitivity for the target pathogen. The detection limit of both assays was 5 fg of C. lindemuthianum genomic DNA. To explore the correlation between the lesion area and the DNA amount of C. lindemuthianum in bean seed, seeds of the navy bean cultivar Navigator with lesions of different sizes, as well as symptomless seeds, were used in both real‐time PCR assays.     

A nested multiplex PCR for species-specific identification and detection of Botryosphaeriaceae species on mango

Lasiodiplodia theobromae (Pat.) Griff. & Maubl, Neofusicoccum parvum Pennycook & Samuels, N. mangiferae Syd. & P. Syd., and Fusicoccum aesculi Corda, all anamorphs of Botryosphaeriaceae species, are the causal agents of mango stem-end rot and fruit rot in Taiwan. Identification of these fungal species based on morphology has not been easy due to their extensive plasticity for some of the morphological characters. To aid reliable identification of Botryosphaeriaceae species associated with mango fruits, four pairs of species-specific primers were designed according to sequences of the ribosomal internal transcribed spacers (ITS), and a rapid method was established based on nested multiplex polymerase chain reaction (PCR) in this study. To perform the analysis, PCR was first run with ITS1 and ITS4 as the primers, followed by a second PCR with the addition of all four sets of species-specific primers. With this method, a low limit of 100?fg^-1?pg of purified fungal DNA was detectable. It could also successfully detect L. theobromae, N. parvum, N. mangiferae and F. aesculi in total DNA extracted from inoculated mango fruits. This assay provides a rapid and sensitive method for the identification of Botryosphaeriaceae species and diagnosis of mango fruit rot and stem-end rot as well.     

Detection of Meloidogyne incognita and Pochonia chlamydosporia by fluorogenic molecular probes*

Fluorescent molecular probes were applied for detection of the plant parasitic nematode Meloidogyne incognita and the nematode‐egg parasitic fungus Pochonia chlamydosporia var. chlamydosporia. A region in the M. incognita rDNA including ITS2 was selected for amplification and recognition with a real‐time PCR assay, based on a combination of three specific motifs, each recognized by a specific fluorescent probe. Similarly, a Scorpion probe was designed for the RT‐PCR quantification of P. c. chlamydosporia. For this purpose, the ITS‐2 rDNA gene of the fungus was sequenced from a number of Italian isolates. A conserved region unique for P. c. chlamydosporia found in the ITS‐2 rDNA gene was used. The probes allowed recognition of single juveniles of M. incognita and of the mycelium‐ or soil‐extracted fungal DNA. The potentialities of the detection procedures are discussed.     

Phenotypic and genetic characterization of Colletotrichum isolates from Belgian strawberry fields

Colletotrichum isolates (457) were collected from strawberry plant tissues with and without typical anthracnose symptoms and from symptomless weeds in 19 Belgian strawberry fields. The isolates were characterized based on genetic, morphological and pathological features. Isolates were classified according to rDNA‐ITS sequencing: 97% of 211 representative isolates were C. acutatum, 2%C. gloeosporioides and 1%C. coccodes. The C. acutatum isolates belonged to the intraspecific groups A2 (33%), A3 (5%), A4 (50%), A5 (3%) and A7 (6%). Differences in spore morphology, growth rate and colony colour of a selection of 146 isolates confirmed the genetic grouping. Multiple Colletotrichum genotypes were detected in the same field. There was no association between the most common genotypes and geographic origin, presence or absence of symptoms, nor plant species or plant part. Representative Belgian Colletotrichum isolates were used in pathogenicity tests, together with European and American reference isolates. The C. acutatum A2 and the Belgian C. gloeosporioides isolates were the most aggressive on fruits, followed by C. acutatum A3, A4, A5, A7 and C. coccodes isolates. When inoculated into crowns, C. acutatum A2, A5 and American C. gloeosporioides isolates were the most aggressive, followed by C. acutatum A3 isolates. The A4 and A7 isolates and all European C. gloeosporioides isolates were non‐pathogenic on crowns. These data indicate that an unusually diverse Colletotrichum population is present in Belgium. The traditional differentiation between C. acutatum and C. gloeosporioides as causal agents of fruit and crown rot, respectively, proved not to be valid in Belgian strawberry fields.     

Characterization of fungal pathogens (Diaporthe angelicae and D. eres) responsible for umbel browning and stem necrosis on carrot in France

A collection of 102 Diaporthe isolates was compiled from lesions on carrot, parsley and wild Apiaceae species in France from 2010 to 2014. Molecular typing based on ITS rDNA sequences resulted in the identification of 85 D. angelicae and 17 D. eres isolates. Based on sequences of the 3′ part of the IGS rDNA, intraspecific variability was analysed for 17 D. angelicae and 13 D. eres isolates from diverse plant species, locations in France, and plant tissues. The genetic diversity was greater for D. angelicae isolates than D. eres isolates. In vitro sensitivity of five D. angelicae and four D. eres isolates to each of nine fungicides was similar for isolates of both species, with a marked variation in fungicide sensitivity depending on the active ingredient. To assess the pathogenicity of D. angelicae and D. eres isolates on carrot, one isolate of each species was inoculated onto umbels in a controlled environment. Typical lesions were observed for both isolates. Carrot crop debris collected from a seed production field in France and placed in controlled conditions produced perithecia and ascospores typical of Diaporthe, that were further characterized molecularly as belonging to D. angelicae. Detection of Diaporthe species on seed lots from three carrot production fields in France was investigated. Both species were detected on seeds by conventional PCR assay, with a greater frequency for D. angelicae than D. eres (67% vs 33%, respectively). Overall, the results highlighted that umbel browning in carrot seed crops in France was mainly caused by D. angelicae.     

Identification,potential inoculum sources and pathogenicity of botryosphaeriaceous species associated with grapevine dieback disease in New Zealand

This study investigated the prevalence and identity of botryosphaeriaceous dieback pathogens in necrotic grapevines tissues in New Zealand vineyards, and other woody hosts growing nearby. The presumptive identities of the isolates by conidial and cultural morphology were confirmed with ITS sequence data as Neofusicoccum australe, N. luteum, N. parvum and Diplodia seriata. They were isolated predominantly from necrotic stems of grapevine and other hosts, but also from leaves, flowers and wood debris of grapevines. Inoculation with conidia and mycelium of multiple isolates of each species onto excised and attached green shoots and trunks of five grapevine varieties, Cabernet sauvignon, Chardonnay, Pinot noir, Riesling, and Sauvignon blanc, showed that all varieties became infected to a similar extent. All species except D. seriata were pathogenic, irrespective of the host source, with N. luteum being the most and D. mutila the least pathogenic (P < 0.05). On trunks, N. parvum caused cankers and the other pathogenic species caused die-back when the inoculated vines became winter-dormant. Conidia were produced from green shoot lesions and die-back wood, which indicates potential inoculum sources for vineyard infection.