Uwe Joachim Nebert ‐ 70 Jahre

Esca is a very important and destructive disease of grapevine in many grapevine producing countries. Several groups of fungi have been reported from grapevines with esca disease symptoms; however, Phaeoacremonium species are the main hyphomycete fungi involved in this disease. In recent years, esca disease symptoms with consequent decline disease have frequently been observed in vineyards of north-western Iran. Nevertheless, the involvement of Phaeoacremonium spp. with esca disease symptoms in this region remains unknown. During 2008–2010 growing seasons, wood samples were collected from vines showing typical esca disease symptoms such as interveinal leaf chlorosis with subsequent necrosis and various types of internal wood deterioration in north-western parts of Iran. A total of 44 Phaeoacremonium-like hyphomycetes were recovered from sampled materials. Fungal isolates were subjected to tentative morphological identification and were further characterised by using sequenced data from ITS-rDNA and β-tublin gene. Phylogeny inferred using sequence data from ITS-rDNA region and β-tublin gene revealed that Phaeoacremonium aleophilum and P. mortoniae together with Sarocladium strictum (syn. Acremonium strictum) are associated with esca disease symptoms in this region. The pathogenicity of Phaeoacremonium spp. and Sarocladium strictum were verified by the inoculation of cutting root and stems under greenhouse conditions. Isolates of P. aleophilum and P. mortoniae were the most virulent based on the length of vascular necrosis on woody stems that followed by Sarocladium strictum. This is first study on the pathogenicity of Sarocladium strictum on grapevine worldwide and the first report on the occurrence of P. mortoniae on grapevines in north-western Iran.     

Major changes in grapevine wood microbiota are associated with the onset of esca,a devastating trunk disease

Esca, a major grapevine trunk disease in old grapevines, is associated with the colonization of woody tissues by a broad range of plant pathogenic fungi. To identify which fungal and bacterial species are involved in the onset of this disease, we analysed the microbiota from woody tissues of young (10-year-old) grapevines at an early stage of esca. Using meta-barcoding, 515 fungal and 403 bacterial operational taxonomic units (OTUs) were identified in woody tissues. In situ hybridization showed that these fungi and bacteria co-inhabited in grapevine woody tissues. In non-necrotic woody tissues, fungal and bacterial microbiota varied according to organs and seasons but not diseased plant status. Phaeomoniella chlamydospora, involved in the Grapevine trunk disease, was the most abundant species in non-necrotic tissues from healthy plants, suggesting a possible non-pathogenic endophytic behaviour. Most diseased plants (70%) displayed cordons, with their central white-rot necrosis colonized essentially by two plant pathogenic fungi (Fomitiporia mediterranea: 60%–90% and P. chlamydospora: 5%–15%) and by a few bacterial taxa (Sphingomonas spp. and Mycobacterium spp.). The occurrence of a specific association of fungal and bacterial species in cordons from young grapevines expressing esca-foliar symptoms strongly suggests that that microbiota is involved in the onset of this complex disease.     

Characterization of Cylindrocarpon liriodendri Associated with Black Foot Disease of Grapevine in Iran

Eight Cylindrocarpon isolates recovered from the trunk bases of 10-year-old grapevines showing decline symptoms from two vineyards in Bavanat (Fars province, south-western Iran) were studied. Based on phenotypical characteristics, mating experiments and molecular data, they were identified as Cylindrocarpon liriodendri. Pathogenicity was confirmed with selected isolates inoculated into 8-month-old dormant rooted cuttings of grapevine rootstock cv. 110 Richter. This is the first report of C. liriodendri causing black foot disease of grapevines in Iran.     

Uneven Distribution of Mating‐Type Alleles Among Togninia minima Isolates,One of the Causal Agents of Leaf Stripe Disease on Grapevines in Northwest Iran

Togninia minima is the main fungal species associated with grapevine leaf stripe disease worldwide. This species is mainly known from its asexual state in nature; nevertheless, a biallelic heterothallic mating strategy has been confirmed for this species based on in vitro crossing studies. There are no data available on the incidence of an active sexual cycle within the populations of this species in many grapevine‐producing countries as well as Iran. The possibility of a clandestine sexual cycle within the Iranian isolates of T. minima was evaluated by analysing the distribution and frequency of the mating‐type alleles on a microspatial and a macrogeographical scales. Towards this aim, a total of 90 T. minima isolates were recovered from grapevines with esca disease from the vineyards in north and north‐western Iran. A multiplex PCR method previously designed by authors was applied for simultaneous identification and determination of the mating‐type alleles in T. minima populations. The results on the screening of mating‐type alleles using multiplex PCR method revealed the mating‐type identity of 77 isolates as Mat1‐2 and 23 isolates as Mat1‐1. Our results showed that both Mat1‐1 and Mat1‐2 isolates are present in a single vineyard and even on single vines. The distribution of mating‐type alleles in the sampled area skewed from the 1 : 1 ratio (77 : 23); however, co‐occurrence of both mating types in a single vineyard and even on single vines is suggestive for the presence of an active sexual cycle for T. minima in north‐western Iran.     

Collophora hispanica,a New Pathogen and Potential Threat to the Almond Industry in Iran

Trunk diseases are potential threats for almond productivity and longevity worldwide, including Iran. In a recent survey on fungal species associated with trunk diseases of almonds in north‐western Iran, Collophora isolates (tentatively identified as Collophora hispanica) were recovered with high frequency from wood samples with internal necrosis and brown to black vascular streaking of almond trees showing symptoms of decline. However, the pathogenic potential of Collophora isolates on almond trees in Iran remains unproven. In this study, the identity of the isolates was further confirmed as C. hispanica based on a combination of morphological data and sequence data of ITS‐rDNA region, and pathogenicity of C. hispanica isolates on almond was evaluated using excised shoot method and in greenhouse experiments. Collophora hispanica isolates induced lesions statistically different from the control, in both excised shoot method and greenhouse assays. Significant differences were observed among the isolates in the length of the lesion induced on wood. Collophora hispanica should be considered as the main trunk pathogens of almond trees in north‐western region of Iran. The distribution and host range of this new pathogen on almond remains to be studied.     

Isolation,molecular characterization and virome analysis of culturable wood fungal endophytes in esca symptomatic and asymptomatic grapevine plants

Europe is the world largest grape producer, but in recent years, the report of diseases due to infection by grapevine trunk pathogens (GTPs) is becoming one of the main constraints for viticulture. Among grapevine trunk diseases (GTDs), esca syndrome is one of the most complex, characterized by simultaneous infection of several fungi, which leads to important reduction in yield and quality. Previous characterization of fungal isolates associated with esca syndrome leads to the recognition of at least three important players: Phaeocremonium minimum, Phaemoniella chlamydospora and Fomitiporia mediterranea. Here we isolated and characterized molecularly fungal endophytes directly from field wood tissues of plants showing or not esca symptoms. In addition, to better characterize such collection, a deep RNA sequencing (100 M reads in paired-end) to screen for mycovirus presence was performed. Thirty-nine viral genomes were detected, 38 of which were putative new viral species; some of these viruses infected GTPs, including P. minimum and F. mediterranea. In this work, we reported for the first time a curated collection of grapevine fungal endophytes identifying the associated mycoviruses some of which could be employed in future biotechnological exploitation as biological control agents for sustainable plant protection.     

A biomarker for the identification of four <Emphasis Type="Italic">Phaeoacremonium</Emphasis> species using the β-tubulin gene as the target sequence

A real-time polymerase chain reaction (PCR) was developed for the rapid detection and identification of Phaeoacremonium species, the fungi associated with severe diseases in grapevines. A degenerate primer pair (F2bt–R1bt) with homology to the β-tubulin gene was designed to be used in the amplification of 11 species of Phaeoacremonium. Four species-specific probes labelled with three different fluorescent dyes were designed to be used with the degenerate primers in a real-time PCR for the identification of Phaeoacremonium aleophilum, P. parasiticum, P. viticola and P. mortoniae. Combinations of two probes in a duplex real-time PCR allowed to detect and identify a mixture of Phaeoacremonium species and cross-amplifications were not detected. This method was applied to detect Phaeoacremonium species in eight wood fragments from grapevine plants naturally infected, and results were compared with those obtained with nested PCR and culturing on growth media. Real-time PCR detected Phaeoacremonium in 100% of the analysed fragments, whereas nested PCR did only in the 62% of them and requiring subsequent restriction fragment-length polymorphism analysis to identify the species. This method is a sensitive tool to detect and identify Phaeoacremonium species in infected grapevine wood. Real-time PCR assay defined here can be used in a plant nursery program to identify pathogen-free plants in order to manage Petri disease of grapevines.     

Weed control and cover crop management affect mycorrhizal colonization of grapevine roots and arbuscular mycorrhizal fungal spore populations in a California vineyard

Arbuscular mycorrhizal (AM) fungi naturally colonize grapevines in California vineyards. Weed control and cover cropping may affect AM fungi directly, through destruction of extraradical hyphae by soil disruption, or indirectly, through effects on populations of mycorrhizal weeds and cover crops. We examined the effects of weed control (cultivation, post-emergence herbicides, pre-emergence herbicides) and cover crops (Secale cereale cv. Merced rye, × Triticosecale cv.Trios 102) on AM fungi in a Central Coast vineyard. Seasonal changes in grapevine mycorrhizal colonization differed among weed control treatments, but did not correspond with seasonal changes in total weed frequency. Differences in grapevine colonization among weed control treatments may be due to differences in mycorrhizal status and/or AM fungal species composition among dominant weed species. Cover crops had no effect on grapevine mycorrhizal colonization, despite higher spring spore populations in cover cropped middles compared to bare middles. Cover crops were mycorrhizal and shared four AM fungal species (Glomus aggregatum, G. etunicatum, G. mosseae, G. scintillans) in common with grapevines. Lack of contact between grapevine roots and cover crop roots may have prevented grapevines from accessing higher spore populations in the middles.     

Vector activity of Hyalesthes obsoletus living on nettles and transmitting a stolbur phytoplasma to grapevines: a case study

We report a case study on the vector activity of a Hyalesthes obsoletus (Hemiptera: Cixiidae) population living on nettle plants (Urtica dioica) and transmitting a stolbur phytoplasma (Sp) to grapevines (Vitis vinifera). The research was conducted in a site that included a vineyard bordered with a large fallow area where nettles were the predominant plant species together with sparse old grapevines. Nettles hosted a high population of H. obsoletus. By using transparent sticky traps to sample adults, we observed that the daily flight activity of males and females to grapevines in the fallow was unimodal peaking between 15 and 21 h in the day. Adults were unable of great dispersion into the vineyard and the pattern of insect captures inside the planting reflected the pattern of Sp‐infected grapevines in the late autumn. When insects were forced to feed on grapevine cuttings for transmission assays, survival of H. obsoletus decreased after 24–48 h. The scarce propensity of the vector to move into the vineyard and feed on grapevines was counterbalanced by the rapidity of H. obsoletus to inoculate Sp to grapevines (estimated minimum inoculation access period ranged from 3 to 6 h) and a relative high incidence of Sp in the population of H. obsoletus that ranged between 20% and 30% of sampled insects as shown by a polymerase chain reaction–based procedure. Characterisation of Sp by restriction fragment length polymorphism analysis of nonribosomal phytoplasma DNA showed the occurrence of an Sp strain known to infect H. obsoletus associated to nettles and grapevines in Germany.     

Detection of Grapevine Viruses in Poland

During a 3‐year study, grapevines from 23 vineyards in Poland were surveyed for virus diseases and tested to determine the prevalence of the most economically important viruses by RT‐PCR. The rate of positive samples was 2.2% for grapevine leafroll‐associated virus 1 (GLRaV‐1), 1.9% for grapevine leafroll‐associated virus 2 (GLRaV‐2), 1.5% grapevine leafroll‐associated virus 3 (GLRaV‐3), 1.9% for grapevine virus A (GVA), 0.2% for grapevine virus B (GVB), 0.2% for grapevine virus E (GVE), 0.65% for grapevine fanleaf virus (GFLV), 20.4% for grapevine fleck virus (GFkV) and 71.9% for grapevine rupestris stem pitting‐associated virus (GRSPaV). These viruses were found to occur as single or mixed infections of different combinations in individual grapevines. The overall viral infection rate in the surveyed grapevines was 82.6%. GRSPaV is the most widely distributed virus of all the viruses currently detected in the region. DNA sequencing confirmed the identification of the viruses in selected samples, and analysis indicated that the Polish isolates shared a close molecular identity with the corresponding isolates in GenBank. To our knowledge, this is the first detection of GLRaV‐1, ‐2, ‐3, GVA, GVB, GVE, GFLV, GFkV and GRSPaV in Poland.     

Phenotypic and molecular characterisation of Colletotrichum acutatum,the causal agent of anthracnose disease on Cornus mas in Iran

Cornus mas L. (Cornaceae), known as European Corneal, is native to south Europe and south-west Asia. This deciduous shrub with edible and medicinal fruits grows wild in Arasbaran forests located in Azerbaijan and Gazvin regions in Northern Iran. A new leaf spot and fruit rot disease have recently been observed on this host in Arasbaran forests located in north-west of Iran. Leaves and fruit samples were collected from heavily infected Cornus mas trees in this region during October 2011. The causal agent of the diseases was identified as Colletotrichum acutatum based on morphological and cultural characteristics. The identity of the species was further confirmed using sequence data from ITS-rDNA region. A phylogeny inferred using sequence data from ITS-rDNA region placed our isolate together with other C. acutatum from host plant species in GenBank. Pathogenicity of C. acutatum was confirmed by the inoculation of C. mas fruits in laboratory condition. The life cycle, distribution and the impact of disease on crop yield remain to be studied.     

PCR-Based Strategy To Detect and Identify Species of Phaeoacremonium Causing Grapevine Diseases

Species of Phaeoacremonium (especially Phaeoacremonium aleophilum) are associated with two severe diseases in grapevines, Petri disease in young plants and Esca disease in adult plants. Phaeoacremonium species grow slowly on culture medium, and it is difficult to identify these species on the basis of morphological characteristics. Primers Pm1 and Pm2 were designed in the ribosomal DNA internal transcribed spacer (ITS) regions ITS1 and ITS2, respectively. They yielded a single amplicon of 415 bp for nine species of Phaeoacremonium that may occur in grapevines. A nested PCR (using general fungal primers ITS1F/ITS4 in the primary reaction) was developed to detect Phaeoacremonium directly in grapevine wood. Molecular detection was more sensitive than the traditional method of culturing in growth medium was. Identification of Phaeoacremonium species was achieved by digesting the PCR-amplified fragment with the restriction enzymes BssKI, EcoO109I, and HhaI. It was possible to distinguish these species by their restriction fragment length polymorphism patterns, except for Phaeoacremonium viticola and Phaeoacremonium angustius, which had 100% similarity in their ITS region sequences. A species-specific PCR amplification of the partial β-tubulin gene using the primer pair Pbr4_1/T1 and Pbr8/T1 was necessary to differentiate P. angustius from P. viticola, respectively. An easy and fast protocol was developed to detect and identify species of Phaeoacremonium in a few hours. Primers defined here can be used in a plant nursery sanitation program to produce plants free of Phaeoacremonium spp. Use of healthy grapevine plants in new plantations is the most effective measure to manage Petri disease.     

Morphological and molecular characterization of Diplodia seriata,the causal agent of canker and twig dieback disease on mulberry in Iran

Mulberries are cultivated for different purposes: for feeding larvae of silkworm, Bombyx mori, as fresh and dry food resources, in wood instrument industry, in pharmaceutical industry and as outdoor ornamental trees in Iran. In recent years, twig and branch canker disease symptoms have been noticed on mulberry trees in northwestern parts of Iran. Diplodia isolates were repeatedly recovered from symptomatic tissues. Based on cultural and morphological features, the isolates were identified as Diplodia seriata. The identity of the isolates was further confirmed using sequence data from internal transcribed spacer (ITS)-rDNA and ef-1α gene. A phylogenetic analysis using ITS sequence data clustered the isolates obtained in this study together with known Diplodia seriata isolates of other woody hosts from GenBank. Inoculation studies carried out on white mulberry twigs using an excised shoot method revealed that the isolates are pathogenic on this host. D. seriata have been reported from other woody host plant species such as Juglans nigra and Vitis vinifera in Iran, however, to the best of our knowledge, the occurrence of D. seriata on mulberry trees is new for Iran. The distribution and reaction of different Morus spp. to D. seriata remain to be studied.     

Bacteria associated with wood tissues of Esca-diseased grapevines: functional diversity and synergy with Fomitiporia mediterranea to degrade wood components

Fungi are considered to cause grapevine trunk diseases such as esca that result in wood degradation. For instance, the basidiomycete Fomitiporia mediterranea (Fmed) is overabundant in white rot, a key type of wood-necrosis associated with esca. However, many bacteria colonize the grapevine wood too, including the white rot. In this study, we hypothesized that bacteria colonizing grapevine wood interact, possibly synergistically, with Fmed and enhance the fungal ability to degrade wood. We isolated 237 bacterial strains from esca-affected grapevine wood. Most of them belonged to the families Xanthomonadaceae and Pseudomonadaceae. Some bacterial strains that degrade grapevine-wood components such as cellulose and hemicellulose did not inhibit Fmed growth in vitro. We proved that the fungal ability to degrade wood can be strongly influenced by bacteria inhabiting the wood. This was shown with a cellulolytic and xylanolytic strain of the Paenibacillus genus, which displays synergistic interaction with Fmed by enhancing the degradation of wood structures. Genome analysis of this Paenibacillus strain revealed several gene clusters such as those involved in the expression of carbohydrate-active enzymes, xylose utilization and vitamin metabolism. In addition, certain other genetic characteristics of the strain allow it to thrive as an endophyte in grapevine and influence the wood degradation by Fmed. This suggests that there might exist a synergistic interaction between the fungus Fmed and the bacterial strain mentioned above, enhancing grapevine wood degradation. Further step would be to point out its occurrence in mature grapevines to promote esca disease development.     

Seasonal Migration of Apolygus spinolae (Hemiptera: Miridae) between Grapevines and Herbaceous Plants

This study was conducted to verify the seasonal migration of Apolygus spinolae (Meyer-Dür) between grapevines and herbaceous plants. Overwintering eggs were hidden in the hair layer under grapevine bud scales. A. spinolae adults were captured on sticky traps in the grapevine yard from late spring to early summer, dwindled through the summer, and captured again in late fall. However, adults were observed from early summer in herbaceous plant fields. A. spinolae adults were abundant during the summer of July and August in the herbaceous field, and thereafter its density decreased through fall. A few or no A. spinolae was found on mesh-netted grapevines after the installation year of the mesh-net, which indicated that A. spinolae adults migrating to lay overwintering eggs during the autumn could not land at the grapevines because of the mesh-net. Damaged shoots by A. spinolae were concentrated near the edge of grapevine yards bordering the herbaceous plants. This distribution pattern of shoot damage was believed to be related to an oviposition behavior of A spinolae, reflecting that adults migrating from herbaceous plants lay eggs more frequently in grapevines adjacent to the summer host plants. Seasonal occurrence of A. spinolae in grapevine yards was suggested as follows: A. spinolae overwinter as eggs in dormant buds on grapevines and hatch in the spring. Nymphs feed on grapevines then develop to adults (spring population), and migrate to herbaceous plants. A. spimolae spends the summer on the herbaceous hosts (summer population). Then, adults migrate back to grapevines in late autumn and lay overwintering eggs.     

Effectiveness of Natural Antifungal Compounds in Controlling Infection by Grapevine Trunk Disease Pathogens through Pruning Wounds

Grapevine trunk fungal pathogens, such as Diplodia seriata and Phaeomoniella chlamydospora, can infect plants through pruning wounds. They cause grapevine trunk diseases and are involved in grapevine decline. Accordingly, the protection of pruning wounds is crucial for the management of grapevine trunk diseases. The efficacy of different natural antifungals in inhibiting the growth of several fungi causing grapevine trunk diseases was evaluated in vitro. The fungi showing greater in vitro efficacy were tested on autoclaved grape wood assays against D. seriata and P. chlamydospora. Based on results from these assays, chitosan oligosaccharide, vanillin, and garlic extract were selected for further evaluation on pruning wounds inoculated with D. seriata and P. chlamydospora in field trials. A significant decrease in plant mortality was observed after 2 years of growth in the plants treated with the different natural antifungals compared to the mortality rate observed in infected plants that were not treated with antifungals. Also, the infection rate for the inoculated pathogens was significantly reduced in plants treated with the selected natural antifungals. Therefore, natural antifungals represent a promising alternative for disease control and could provide significant economic benefits for the grape-growing industry.     

Gibberellins and Abscisic Acid Promote Carbon Allocation in Roots and Berries of Grapevines

Carbon allocation within grapevines may affect berry growth and development. The plant hormones gibberellins (GAs) and abscisic acid (ABA) control various processes across the plant life and both have been involved in assimilate production and transport in different species. Hence, this work examined the distribution of sugars (sucrose, fructose, and glucose) and starch in grapevines at veraison after foliar applications of GA₃, ABA, and an inhibitor of GA biosynthesis, paclobutrazol (PBZ). The results demonstrated that GA₃ increased total grapevine mass, with carbon allocated to the whole grapevine (as structural and soluble carbohydrates). Both GA₃ and ABA increased monosaccharide (glucose and fructose) levels in berries (up to tenfold) and roots (up to threefold). However, GA₃ increased the net carbon fixation whereas ABA did not. PBZ diminished most growth parameters except grapevine mass, and allocated more carbohydrates to roots (up to threefold more sucrose and starch). Such results indicate that GAs promote net carbon fixation and transport, whereas ABA as a stress signal only enhances sugar transport; notwithstanding the two hormones promoted carbon allocation toward roots and berries.     

Di Alcuni Caratteri Differenziali Nelle Plantule di “ Cuscuta Epithymum „ Mürr. e di “ Cuscuta Pentagona „ Eng.

Endophytes are micro-organisms that colonize the internal tissues of plants without inducing signs of negative effects and that can provide benefits to plant health and yield. In the present work, the culturable bacterial endophyte community, colonizing vegetative organs of grapevine, was isolated from surface-sterilized plant tissues and characterized by molecular methods. From roots, shoots and leaves of Vitis vinifera “Glera”, located in six different vineyards throughout the Conegliano-Valdobbiadene DOCG area (Veneto, Italy), 381 culturable strains were successfully isolated; amplified ribosomal DNA restriction analysis and nucleotide sequencing showed that approximately 30% of the endophyte community belonged to the genus Bacillus, which was the most represented; other genera such as Staphylococcus, Microbacterium, Paenibacillus, Curtobacterium, Stenotrophomonas, Variovorax, Micrococcus and Agrococcus were identified. Endophyte community composition within each vine was different in respect to other endophyte populations living in grapevine plants coming from different vineyards; moreover, the bacterial composition changed depending on the season of sampling. The above data highlight the great diversity of culturable bacterial species inhabiting Glera grapevines and open the way for a characterization and selection of strains that could potentially be used to improve the vineyard management for plant growth and yield, plant responses to stresses, biocontrol and biofertilization.