Association between genetic variability and titre of Grapevine Pinot gris virus with disease symptoms

The present work was carried out in order to verify the possible association between a new grapevine disease, characterized by leaf mottling and deformation, and the genetic variability and concentration of Grapevine Pinot gris virus (GPGV), a recently identified virus tentatively associated with the pathology. After vineyard surveys and the establishment of real‐time qPCR assays, characterization of GPGV isolates and evaluation of GPGV titre were assessed in more than 100 samples of grapevine Glera, collected from plants regardless of whether or not they showed the symptomatology. Results showed that there was an important association between the GPGV variants and manifestation of the symptoms, and that grapevines with symptoms harboured significantly higher GPGV titre than symptomless vines. Moreover, an interesting relationship among the phylogenetic clustering of the isolates originating from plants with symptoms and some epidemiological characteristics of the disease was found. The current study confirmed the role of GPGV in the emergent disease characterized by grapevine leaf mottling and deformation.     

High genotypic and virulence diversity in Ilyonectria liriodendri isolates associated with black foot disease in New Zealand vineyards

A total of 57 Ilyonectria liriodendri isolates were identified by a combination of species‐specific PCR and DNA sequencing from a collection of 174 Ilyonectria‐like isolates recovered from 101 diseased grapevine samples. These samples were representative of the national vineyard, comprising material contributed by 49 grape growers across seven grape growing areas. This species was predominant, representing 33% of the recovered isolates, and has been reported as a major pathogen of grapevines in other countries. The genetic diversity of the 57 New Zealand isolates was compared to that of isolates from Australia and South Africa using universally primed polymerase chain reaction (UP‐PCR). A total of 66 informative loci distinguished 52 genotypes, of which five contained up to four clonal isolates. Four main clades were identified in a neighbour‐joining (NJ) tree. The international isolates (Australia and South Africa) were placed in a clade that did not include New Zealand isolates. There was a high level of intra‐ and inter‐vineyard genetic variation indicating the free movement of isolates between regions. A subset of nine isolates from different branches of the NJ tree produced two vegetative compatibility groups and hyphal fusion was observed between non‐self pairings. Pathogenicity tests using isolates from different genetic groups inoculated onto either detached roots or 1‐year‐old potted vines showed variability in virulence; however, no correlations were detected.     

Aetiology of anthracnose on grapevine shoots in Brazil

Anthracnose is an important disease in vineyards in south and southeast Brazil, the main grape‐producing regions in the country. This study aimed to identify the causal agents of grapevine anthracnose in Brazil through multilocus phylogenetic analyses, morphological characterization and pathogenicity tests. Thirty‐nine Elsinoë ampelina and 13 Colletotrichum spp. isolates were obtained from leaves, stems and berries with anthracnose symptoms collected in 38 vineyards in southern and southeastern Brazil. For E. ampelina isolates, the internal transcribed spacer (ITS), histone H3 (HIS3) and elongation factor 1‐α (TEF) sequences were analysed. HIS3 was the most informative region with 55 polymorphic sites including deletions and substitutions of bases, enabling the grouping of isolates into five haplotypes. Colonies of E. ampelina showed slow growth, variable colouration and a wrinkled texture on potato dextrose agar. Conidia were cylindrical to oblong with rounded ends, hyaline, aseptate, (3.57–) 5.64 (?6.95) μm long and (2.03–) 2.65 (?3.40) μm wide. Seven species of Colletotrichum were identified: C. siamense, C. gloeosporioides, C. fructicola, C. viniferum, C. nymphaeae, C. truncatum and C. cliviae, with a wide variation in colony and conidium morphology. Only E. ampelina caused anthracnose symptoms on leaves, tendrils and stems of Vitis vinifera and V. labrusca. High disease severity and a negative correlation between disease severity and shoot dry weight were observed only when relative humidity was above 95%. In this study, only E. ampelina caused anthracnose symptoms on grapevine shoots in Brazil.     

Maßnahmen zur Eindämmung des Brennnesseltyps der Schwarzholzkrankheit bei Weinreben (Vitis vinifera)

Since 2003 incidence of Bois noir disease (Stolbur Type I) in the area of Baden-Württemberg has rapidly increased. Hylasthes obsoletus, known vector cicada transmitting Bois noir disease from stinging nettle to grapevine, was found on stinging nettles located at vineyard walls or vineyard tracks. Phytoplasma of Stolbur Type I (host plant: stinging nettles) were also detected within samples of Hylasthes obsoletus. Incidence of Bois noir disease could be strongly reduced by either not mowing or destroying stinging nettles (host plant of Hyalesthes obsoletus) between June and August, the flight period of Hylasthes obsoletus, or by eradication of stinging nettles in winter. Fighting stinging nettles between November and March with Glyphosate resulted in a dramatic decrease of vector cicada. Pruning experiments of the extremely sensitive grape variety Lemberger and the variety Trollinger showed that survival of grape vines significantly improved compared to normal pruning in winter if the vines were cut off 10?cm above ground immediately after detection of the first symptoms. In contrast, cutting off vines 10?cm above ground in summer had no effect on survival rates of 40 year old grape vines of the variety Pinot noir. Furthermore, most of these old grapevines did not survive this dramatic pruning measure. Generally it is advised to cut off diseased plant material as soon as symptoms become visible.     

Spatial and temporal stolbur population structure in a cv. Chardonnay vineyard according to vmp1 gene characterization

Bois noir is a grapevine disease caused by the stolbur phytoplasma. It is widespread in all European and Mediterranean viticultural areas, and it can induce severe damage to the quality and quantity of production. The recent disease recrudescence has encouraged studies on the use of molecular markers to assess the genetic diversity of stolbur strains. The aim of this study was to evaluate the presence of Bois noir symptoms and to monitor the spatial genetic structure of the stolbur population according to vmp1 genotypes, through 2011 and 2012 in a cv. Chardonnay vineyard. In both years, there were increased vines with symptoms from July to September. The analysis of dispersal indices showed that the spatial distribution was uniform in the vineyard. However, the two‐dimensional contour maps show that Bois noir severity was higher in plants located on the borders than in the central parts of the vineyard. Stolbur population was composed of two prevalent vmp genotypes (V14, V12) across both years, along with other minor haplotypes (V3, V4, V9, V11, V15, V18, in 2011; V3, V18 in 2012). The data indicate that the vmp1 gene is an efficient marker to study the population structure of stolbur phytoplasma, to track the movement of the pathogen, and to identify the inoculum source, which will all serve in the planning of control strategies.     

A mechanistic model simulating ascosporic infections by Erysiphe necator,the powdery mildew fungus of grapevine

A new dynamic model for Erysiphe necator ascosporic infections on grapevine was developed. Between budbreak of vines and the time when the pool of ascospores is depleted, the model uses weather data for calculating, at daily intervals: curve of ascospore maturation; ascospore discharge events and relative proportion of the discharged ascospores; infection periods and their relative infection severity; and progress of latency period and time when secondary infections should begin. The model was validated over a 4‐year period (2005–2008) in 26 vineyards in Italy by comparing model predictions with actual observations of the first seasonal symptoms of powdery mildew. The model showed high sensitivity, specificity and accuracy. Proportions of true and false positive predictions were TPP = 0·94 and FPP = 0·26, respectively. Because a proportion of predicted infection periods did not result in actual disease onset, confidence was higher for prediction of non‐infections than for prediction of infections. Most of the false positive predictions occurred in the earlier growth stages of the host, when the surface area of susceptible tissue may be very small so that the probability that ejected ascospores land on susceptible tissue is low. An equation was then developed to describe the probability that a predicted infection period results in disease onset as a function of the growth stage of vines at the time of prediction. The new model should improve early season powdery mildew management by helping vineyard managers schedule fungicide sprays or schedule the scouting of the vineyard for detection of first disease signs.     

Identification of grapevine marker genes for early,non‐destructive Eutypa lata infection diagnosis

Eutypa lata is the causal agent of eutypa dieback, a highly damaging trunk disease affecting all grape‐growing areas, with currently neither an efficient curative treatment nor an early non‐destructive diagnostic method. The present work was carried out to discover grapevine genes expressed in response to the presence of E. lata that could be useful to develop an early (before visible foliar symptoms) and non‐destructive (using grapevine leaves) diagnostic tool. Microarray analyses were carried out from (i) infected plants showing characteristic E. lata foliar and vascular symptoms and positive pathogen recovery from vascular lesions (S⁺R⁺), (ii) infected plants showing no symptoms (S^?R⁺), and (iii) symptomless plants with negative pathogen recovery (S^?R^?). Vineyard and greenhouse‐grown plants, naturally or artificially infected respectively, and uninoculated controls were characterized and leaf RNA was hybridized with 15k operon grapevine oligonucleotide microarrays. Among the grapevine genes differentially expressed between S^?R⁺ and S^?R^? plants in greenhouse and vineyard conditions, 10 were highlighted as robust candidate genes for diagnosis: seven were specifically involved in response to infection and three were associated with symptom absence. Five were confirmed to be effective diagnostic marker genes usable in a qRT‐PCR‐based test performed on RNA extracted from grapevine leaves cultivated in either greenhouse or vineyard conditions. Furthermore, their expression profiles in response to infection with E. lata or other major grapevine fungi (Erysiphe necator, Plasmopara viticola, Botrytis cinerea) could be distinguished. The usefulness of these genes to develop an early and non‐destructive method for diagnosis of E. lata infection is discussed with regard to the advantages and drawbacks of previous E. lata diagnostic studies.     

The taxonomic position of Xanthomonas ampelina1

The internal taxonomic structure and the suprageneric relations of the causal agent of bacterial blight and canker of grapevine, the misnamed Xanthomonas ampelina, were examined. By studying more than 30 strains of different geographical origin by polyacrylamide gel electrophoresis and DNA: DNA hybridization, we established the homogeneity of X. ampelina. [¹⁴C]-labelled ribosomal RNA (rRNA) was prepared from the type strain of X. ampelina (NCPPB 2217) and hybridized with DNA from different representative strains from the Pseudomonas acidovorans rRNA complex in rRNA superfamily III. X. ampelina was found to be a separate branch in rRNA superfamily III, related at a suprageneric level to P. acidovorans, Alcaligenes paradoxus and the other organisms of the P. acidovorans rRNA complex.     

Recent research progress on Xanthomonas ampelina1

Bacterial blight of grapevine (caused by Xanthomonas ampelina) occurs in Greece, France, Spain, Italy, Turkey, Portugal, USSR and South Africa. It is a serious, chronic vascular disease which is widespread in some areas of grape production, affecting commercially important cultivars. There is evidence that the disease is presently spreading into new areas in some countries. The actual distribution of blight may be much wider, since its symptoms may be confused with those of other diseases. It is therefore essential to confirm the presence of X. ampelina by isolation and identification. X. ampelina is an anomalous member of the genus Xanthomonas which is readily distinguished on the basis of a few tests. The pathogen may also be rapidly detected in grapevine sap using the indirect immunofluorescence technique. The susceptibility of shoots to infections, under Cretan conditions, is highest from November to late January and very low during February and March. It has been found that up to 50% of apparently healthy canes obtained from diseased vineyards were latently infected. Transmission of the pathogen through soil and roots during the control of Daktulosphaira vitifolii or frost damage by flooding has been demonstrated in France. Several of the more widely grown cultivars were found to be very susceptible to the pathogen while no completely resistant cultivar has been detected. Control trials based on four sprays with a number of chemicals were unsuccessful. Control policy should be directed: (a) to preventing spread of the disease, by exclusive use of certified material; (b) to minimizing the disease in affected areas by suitable sanitary, cultural and preventive measures.     

Biological control of sclerotinia stem rot of canola using antagonistic bacteria

Stem rot caused by Sclerotinia sclerotiorum is a major fungal disease of canola worldwide. In Australia the management of stem rot relies primarily on strategic application of synthetic fungicides. In an attempt to find alternative strategies for the management of the disease, 514 naturally occurring bacterial isolates were screened for antagonism to S. sclerotiorum. Antifungal activity against mycelial growth of the fungus was exhibited by three isolates of bacteria. The bacteria were identified as Bacillus cereus (SC‐1 and P‐1) and Bacillus subtilis (W‐67) via 16S rRNA sequencing. In vitro antagonism assays using these isolates resulted in significant inhibition of mycelial elongation and complete inhibition of sclerotial germination by both non‐volatile and volatile metabolites. The antagonistic strains caused a significant reduction in the viability of sclerotia when tested in a greenhouse pot trial with soil collected from the field. Spray treatments of bacterial strains reduced disease incidence and yielded higher control efficacy both on inoculated cotyledons and stems. Application of SC‐1 and W‐67 in the field at 10% flowering stage (growth stage 4·00) of canola demonstrated that control efficacy of SC‐1 was significantly higher in all three trials (over 2 years) when sprayed twice at 7‐day intervals. The greatest control of disease was observed with the fungicide Prosaro^® 420SC or with two applications of SC‐1. The results demonstrated that, in the light of environmental concerns and increasing cost of fungicides, B. cereus SC‐1 may have potential as a biological control agent of sclerotinia stem rot of canola in Australia.     

A yellows disease system with differing principal host plants for the obligatory pathogen and its vector

A stolbur‐type phytoplasma is the putative pathogen of grapevine yellows disease that causes economic damage to vineyards in most growing areas around the world. The pathogen is known to be transmitted to vines by two planthoppers, Hyalesthes obsoletus and Reptalus panzer; the latter is found in Europe but has not yet been observed in Israel. The establishment of a vector–pathogen–plant relationship requires that the pathogen and the vector meet on a shared host plant. This does not happen in the ecosystem examined here, where two different principal host plants for the obligate pathogen and its vector exist: the pathogen is established on vines, while its vector, H. obsoletus, develops on Vitex agnus‐castus. The present study verified that: (i) the vector cannot complete its life cycle on vines; (ii) V. agnus‐castus does not grow in the immediate vicinity of vines, and does not harbour the pathogen; and (iii) the pathogen is not vertically transmitted from mother to offspring. Moreover, in a thorough search of plants in vine growing areas, no other plants were found that host both the vector and the pathogen. However, it was found that the planthopper can acquire the phytoplasma from infected vines. Nonetheless, this does not prove the ability of the planthopper to further transmit the pathogen to vines and does not explain the presence of the vector on the non‐preferred vines. Thus, the enigma of the pathogen–vector–host triangle in this system remains unresolved.     

Role of perithecia as an inoculum source for stem rot type of pepper root rot caused by Fusarium solani f. sp. piperis (teleomorph: Nectria haematococca f. sp. piperis)

Pepper (Piper nigrum L.) root rot caused by Fusarium solani f. sp. piperis (FSP; teleomorph: Nectria haematococca f. sp. piperis) includes two symptom types called root rot (RR) type and stem rot (SR) type. In this study, the temporal and spatial associations between perithecial formation of FSP and development of SR were investigated in naturally infested fields to verify the hypothesis that ascospores from the perithecia are the major inoculum source of the SR type on vines in the field. In surveys of all vines in two neighboring pepper fields every month from December 2005 to November 2006, I mapped the locations of all vines with perithecia and all vines the SR type. The frequency of vines with perithecia increased during April and May, the late rainy season. In June, the early dry season, the number of vines with SR type greatly increased. The vertical range of perithecial formation on the vines extended to 200 cm in height, but was restricted to 30 cm in the dry season in both fields. The join-count statistics showed a significant spatial association between vines with perithecia and vines with SR type in one field (P = 0.042), while no significant spatial association was recognized in another field. The results suggested that ascospores from perithecia of FSP on pepper vines are likely to be one of the main inoculum sources of the SR type of the disease on adjacent vines, but they may not be the exclusive source.     

Root‐knot nematodes do not affect black root rot disease severity on watermelon and bottle gourd

The effects of root‐knot nematodes on black root rot of watermelon and bottle gourd were studied using field surveys and co‐inoculation tests with Meloidogyne incognita (southern root‐knot nematode) and Diaporthe sclerotioides. The results of the field survey suggested that root‐knot nematodes had little effect on either the severity of black root rot or infection with D. sclerotioides. None of the three fields showed a significant positive correlation between disease severity and nematode gall index, with low correlation coefficients. Co‐inoculation experiments under controlled conditions found no significant effect of root‐knot nematodes on black root rot of watermelon and bottle gourd based on area under disease progress curves (AUDPC). These results were supported by the quantities of DNA of the two agents in root tissues because no significant difference was found between dual‐ and single‐inoculation treatments with M. incognita and/or D. sclerotioides. These findings suggest that root‐knot nematodes probably do not affect the infection of watermelon or bottle gourd roots by D. sclerotioides or the incidence of black root rot in these crops caused by this fungus.     

Identification of Berkeleyomyces rouxiae causing black root rot disease on cotton seedlings in New South Wales,Australia

Black root rot disease of cotton seedlings caused by Thielavioposis basicola was first reported in New South Wales (NSW), Australia in 1990. In 2018, T. basicola was reclassified into a new genus Berkeleyomyces, accommodating two closely related species: B. basicola and B. rouxiae. However, species status of cotton-T. basicola in NSW remains unsolved. Ninety-five isolates recovered from black root rot diseased cotton seedlings sampled across NSW in 2017/18 season was subjected to morphological, multigene sequencing (ITS, MCM7, RPB2), and pathogenicity assessments for their species identification. Berkeleyomyces rouxiae was accurately identified as the causal agent of black root rot of cotton.

     

No evidence of transmission of grapevine leafroll-associated viruses by phylloxera (<Emphasis Type="Italic">Daktulosphaira vitifoliae</Emphasis>)

Grapevine leafroll disease is associated with several species of phloem-limited grapevine leafroll-associated viruses (GLRaV), some of which are transmitted by mealybugs and scale insects. The grape phylloxera, Daktulosphaira vitifoliae (Fitch) Biotype A (Hemiptera: Phylloxeridae), is a common vineyard pest that feeds on the phloem of vine roots. There is concern that these insects may transmit one or more GLRaV species, particularly GLRaV-2, a species in the genus Closterovirus. A field survey was performed in vineyards with a high incidence of grapevine leafroll disease and D. vitifoliae was assessed for acquisition of GLRaV. In greenhouse experiments, the ability of D. vitifoliae to transmit GLRaV from infected root sections or vines to co-planted virus-free recipient vines was tested. There were no GLRaV-positive D. vitifoliae in the field survey, nor did D. vitifoliae transmit GLRaV-1, ?2, ?3, or -4LV in greenhouse transmission experiments. Some insects tested positive for GLRaV after feeding on infected source vines in the greenhouse, however there was no evidence of virus transmission to healthy plants. These findings, in combination with the sedentary behaviour of the soil biotype of D. vitifoliae, make it unlikely that D. vitifoliae is a vector of any GLRaV.     

Co‐infection by Botryosphaeriaceae and Ilyonectria spp. fungi during propagation causes decline of young grafted grapevines

Decline of newly planted, grafted grapevines is a serious viticultural problem worldwide. In the Riverina (New South Wales, Australia), characteristic symptoms include low fruit yields, very short shoots and severely stunted roots with black, sunken, necrotic lesions. To determine the cause, roots and wood tissue from affected plants in 20 vineyards (Vitis vinifera cv. Chardonnay grafted to V. champini cv. Ramsey rootstock) were assayed for microbial pathogens. Ilyonectria spp. (I. macrodidyma or I. liriodendra, producers of phytotoxin brefeldin A, BFA, and cause of black foot disease of grapevines) and Botryosphaeriaceae spp. (predominantly Diplodia seriata) were isolated from rootstocks of 100 and 95% of the plants, respectively. Togninia minima and Phaeomoniella chlamydospora (cause of grapevine Petri disease) were isolated from 13 and 7% of affected plants, respectively. All Ramsey rootstock stems of grafted plants sampled from a supplier nursery were infected with Ilyonectria spp. and D. seriata. Diplodia seriata, but not Ilyonectria spp., was also isolated from 25% of canes sampled from the rootstock source block. Root inoculation of potted, disease‐free Chardonnay plants with Ilyonectria isolates from diseased vineyards caused typical disease symptoms, while co‐inoculation with Botryosphaeriaceae spp. increased disease severity. This is the first study to show that a major cause of young grapevine decline can be sequential infection by Botryosphaeriaceae from rootstock cuttings and Ilyonectria spp. from nursery soil. Although the Petri disease fungi were less common in young declining grafted grapevines in the Riverina, they are likely to contribute to the decline of surviving plants as they mature.     

First laboratory confirmation of Xylophilus ampelinus in Slovenia*

Bacterial blight of grapevine is caused by a slow‐growing bacterium Xylophilus ampelinus. It has been suspected to occur in Slovenia on the basis of visual observation of characteristic symptoms in the 1960s. In the present study, symptoms were recorded in an infected vineyard during three consecutive years (2002/2004). Samples from this vineyard were tested by nested‐PCR and isolation of bacteria on media was attempted. In the first year, angular lesions on leaves were highly expressed and an isolate morphologically similar to X. ampelinus was obtained from one sample. It was purified and identified as X. ampelinus using biochemical and nutritional tests, fatty acid analysis, immuno‐fluorescence, nested PCR and partial sequencing of the 16S rRNA gene. The 16S rDNA sequence showed 99–100% homology to known sequences of X. ampelinus strains, including the type strain. Pathogenicity of the isolate was confirmed in tissue‐cultured and potted grapevine plants. In the following two years, symptoms of bacterial blight were only faintly expressed. Using isolation on media and nested‐PCR, 23 and 17 extracts prepared from 10 and 8 grapevines, respectively, were analysed. In 2003, no positive sample was found, but X. ampelinus was again isolated and identified by colony morphology and nested‐PCR in 2004.     

Biological control of botrytis bunch rot in organic wine grapes with the yeast antagonist Candida sake CPA‐1

The aim of this research was to confirm the efficacy of the yeast antagonist Candida sake CPA‐1 in suppressing botrytis bunch rot development, in an organic vineyard under Mediterranean conditions for two seasons, and compare its performance with that of two biologically based products currently registered for botrytis bunch rot control in New Zealand. In 2009, treatments applied were: commercial formulations of Ulocladium oudemansii (BOTRY‐Zen^®) and chitosan (ARMOUR‐Zen^®), C. sake CPA‐1 combined with the fatty acid‐based additive Fungicover^® and combinations of these products. All treatments were applied six times between early flowering and harvest and compared with an unsprayed control. In 2010, the treatments focused on C. sake and Fungicover and the number of applications was reduced from six to four. The population dynamics of U. oudemansii and C. sake were measured and wine quality tests were carried out in both seasons. Disease control achieved by C. sake treatments in 2009 were comparable to those achieved by BOTRY‐Zen and ARMOUR‐Zen. Applications of C. sake plus Fungicover between flowering and harvest significantly (P < 0·05) reduced botrytis bunch rot incidence and severity by 64% and 90%, respectively, compared with the untreated control in 2009, and by 67% and 89%, respectively, in 2010. Treatments did not adversely affect wine quality parameters after treated grapes were processed. Candida sake consistently provided effective control of botrytis bunch rot in grapes under different meteorological and disease pressure conditions, thereby improving its potential for future commercial applications.