Pathogenicity of ten Phaeoacremonium species associated with esca and Petri disease of grapevine

CITATION: Baloyi, M., Mostert, L. & Halleen, F. 2018. Pathogenicity of ten Phaeoacremonium species associated with esca and Petri disease of grapevine. Phytopathologia Mediterranea, 57(3):538-546, doi:10.14601/Phytopathol_Mediterr-23940.The original publication is available at http://www.fupress.netNineteen species of Phaeoacremonium have been associated with grapevines in South Africa, of which only six species have been confirmed as pathogens through pathogenicity tests conducted on field-grown grapevines. This study determined the pathogenic status of ten Phaeoacremonium spp. recently found for the first time on South African grapevines. These were: Pm. australiense, Pm. austroafricanum, Pm. fraxinopennsylvanicum, Pm. griseo-olivaceum, Pm. griseorubrum, Pm. iranianum, Pm. italicum, Pm. prunicolum, Pm. scolyti and Pm. sicilianum. In the pathogenicity tests, Ph. parasiticum was used as the positive control, and sterile water as the negative control. Up to three isolates were used per species, depending on isolate availability. Freshly cut pruning wounds in a 9-yearold Cabernet Sauvignon vineyard in Stellenbosch, South Africa, were inoculated with 200 conidia of each fungus per wound. Inoculated pruning wounds were removed after 18 months, cut longitudinally and lesion lengths were measured. Re-isolation proportions were determined by conducting isolations from inoculated spurs. All the inoculated isolates successfully colonized pruning wounds, and caused lesions that were significantly different from the negative control. All isolates were re-isolated at proportions varying from 28.6 to 85.7%. Phaeoacremonium griseo-olivaceum STE-U 7859 produced the longest lesions (mean = 79.5 mm) and Pm. iranianum STE-U 6998 the shortest (62.0 mm). No statistically significant differences in mean lesion lengths were observed between the inoculated species. There were also no significant differences between isolates of the same species, except in Pm. prunicolum where isolate STE-U 5968 produced longer lesions (mean = 77.3 mm) than STE-U 7857 (62.3 mm). This study confirmed the capabilities of all the tested Phaeoacremonium spp. to infect grapevine pruning wounds and cause lesions. The study also confirmed the importance of pruning wounds as ports of entry by these pathogens into host plants.https://oajournals.fupress.net/index.php/pm/article/view/5790Publisher's versio

Hymenochaetales associated with esca-related wood rots on grapevine with a special emphasis on the status of esca in South African vineyards

Esca disease is a problem on grapevines worldwide. This disease complex is characterised by several external and internal symptoms including foliar tiger-stripe chlorosis and necrosis, dieback, wood necrosis and white rot. The causal organisms of esca are primarily Phaeomoniella chlamydospora, several Phaeoacremonium species and basidiomycete species from the order Hymenochaetales, the latter ones responsible for causing the white rot symptom. Basidiomycete species causing the wood rot symptom of esca differ among grapevine-growing areas worldwide. South African vineyards are unique in having a minimum of ten different basidiomycete taxa from five different genera associated with the esca complex. In general, Hymenochaetales species are associated with white rot on woody plants and there are several species that are economically important to the agricultural and forestry industries. Few Hymenochaetales species have been described from the African continent, though this review is an indication of the previously unknown diversity of these fungi in Southern Africa

Grapevine cultivar variation to pruning wound protection by Trichoderma species against trunk pathogens

Using Trichoderma species to protect grapevine pruning wounds from trunk disease pathogensis one of the options available for managing grapevine trunk diseases. The growth and persistence of Trichoderma species in the pruning wound and the resulting control effect may depend on intrinsic wound factors and hence may vary between cultivars. Cultivar variability to pruning wound protection by Trichoderma species was evaluated in eight wine grape (Cabernet Sauvignon, Chardonnay, Chenin blanc, Colombar, Merlot,Pinotage, Sauvignon blanc and Shiraz) and four table grape (Prime, Red Globe, Thompson Seedless andVictoria) cultivars. Two strains of Trichoderma atroviride (USPP-T1 and USPP-T2) separately or in combinationand Eco 77® a registered pruning wound biocontrol agent based on T. harzianum, were applied to fresh pruning wounds of spur-pruned wine grapevines and cane-pruned table grapevines. Trichoderma spp. and a variety of trunk pathogens, Phaeomoniella chlamydospora and species of Phaeoacremonium, Phomopsis, Botryosphaeriaceae and Diatrypaceae, were isolated from the pruning wounds eight months after treatment. Significant treatment × cultivar interactions (P<0.01) were found in the incidence of Trichoderma spp. in table and wine grapes. Trichoderma incidence varied greatly between cultivars and was less between Trichoderma treatments within the same cultivar. The highest Trichoderma incidence in wine grapes was found in Chenin blanc (71.4–82.5%) and in table grapes in Thompson Seedless (43.5–76.7%). In the remaining winegrape cultivars Trichoderma incidence varied between 20–50% while with all treatments in Chardonnay Trichoderma incidence was less than 24%. In table grapes Trichoderma incidence varied from 20-67% in the other cultivars. Trichoderma reduced the pathogen by between 10.3% in Chardonnay to 66.7% in Cheninblanc. Trichoderma incidence and pathogen reduction were significantly correlated in most cultivars (r> 0.50;P<0.05) though not in Chardonnay (r=0.37; P=0.11), Pinotage (r=-0.12; P=0.62), Sauvignon blanc (r=0.26;P=0.26) and Victoria (r=0.29; P=0.22). It was concluded that the wound protection effect of Trichoderma spp.is also dependent on the Trichoderma-grapevine interaction and is not only due to the suppressive effect of Trichoderma spp. on the pathogens

Histo-pathology study of the growth of Trichoderma harzianum, Phaeomoniella chlamydospora and Eutypa lata on grapevine pruning wounds

Protecting grapevine pruning wounds by inoculating them with Trichoderma spp. can preventinfection from trunk disease pathogens. The growth and interactions of both, the biological control agent Trichoderma spp. and the vine pathogens, are not well understood. Green fluorescent protein (GFP)-labelled Trichoderma harzianum and red fluorescent protein (DsRed)-labelled T. harzianum, were dual-inoculatedwith Phaeomoniella chlamydospora (DsRed) or Eutypa lata (GFP) on fresh pruning wounds of one-year-oldCabernet Sauvignon and Sauvignon blanc shoots. The inoculated fungi were recovered from varying depthswithin the shoots at 30-day-intervals for 90 days. Trichoderma harzianum suppressed the pathogens and grew deeper in the presence of the pathogens than when it was singly inoculated; possibly an indication of pathogen recognition and competitive response. Eutypa lata was completely eliminated from Sauvignon blancin dual-inoculated canes after 90 days. The mycelium of P. chlamydospora (DsRed) grew extensively in thexylem vessels and possibly contributed to vessel occlusion. Phaeomoniella chlamydospora and E. lata caused blockage of the vessels and thickening of the vessel walls. Grapevine wood produced both tyloses and gums (gels) that blocked xylem vessels as a result of infection. A thickening of the cell walls of xylem fibres occurredonly in E. lata-inoculated shoots, indicative of a different mode of pathogenesis from P. chlamydospora

Characterisation of the fungi associated with esca diseased grapevines in South Africa

During the period from 2001 to 2008, grapevines showing foliar and/or internal symptoms of esca were collected from various grape-growing regions in South Africa. Isolations were made from typical internal wood symptoms associated with esca, and fungal isolates were characterized by cultural growth patterns, morphology and phylogenetic inference. The gene regions sequenced included the internal transcribed spacers and the 5.8S rRNA gene (ITS) for the basidiomycetes and Phomopsis isolates, the partial b-tubulin and actin genes for Phaeoacremonium isolates and the partial translation elongation 1-a gene and the ITS for the Botryosphaeriaceae isolates. The fungi identified included Phaeomoniella chlamydospora and six species of Phaeoacremonium including Pm. aleophilum, Pm. alvesii, Pm. parasiticum, Pm.iranianum, Pm. mortoniae and Pm. sicilianum, of which the latter three are reported for the first time in South Africa. The following taxa were also identified: Eutypa lata, Phomopsis viticola, Phomopsis theicola, Diaporthe ambigua, Diplodia seriata, Neofusicoccum australe and N. parvum. The basidiomycete isolates were distributed over ten well supported monophyletic clades among genera of the Hymenochaetales. Two of these clades could be identified as species of Fomitiporia and Phellinus

Effects of «Trichoderma» Treatments on the Occurrence of Decline Pathogens in the Roots and Rootstocks of Nursery Grapevines

The growth-stimulating attributes of Trichoderma treatments (dips, soil amendments and drenches with Trichoderma products containing propagules of selected strains of Trichoderma harzianum) in grapevine nurseries, and their effect on the occurrence of fungi in roots and rootstocks of nursery grapevines, in particular fungi causing Petri disease (Phaeomoniella chlamydospora and Phaeoacremonium spp.) and black foot rot (Cylindrocarpon spp.), were compared with quintozene/procymidone treated (standard) vines. Early shoot growth of Trichoderma treated vines was visibly better than that of the control vines. Eight months after planting, at uprooting, percentage take and shoot mass of Trichoderma and standard treated vines were similar, but total root mass was significantly higher for Trichoderma treated vines. Low percentages of Cylindrocarpon spp. were isolated from the rootstocks of treated and untreated vines, while less Petri disease fungi were isolated from rootstocks of Trichoderma treated vines. Markedly fewer fungi were also isolated from the roots of Trichoderma treated vines. Incidences of Petri disease fungi in roots of Trichoderma and standard treated vines were similar, but fewer Cylindrocarpon spp. were isolated from Trichoderma treated vines. These results indicate the potential of Trichoderma treatments in grapevine nurseries for the production of stronger vines with lower Phaeomoniella/Phaeoacremonium and Cylindrocarpon infection levels

Occurrence fungi causing black foot on young grapevines and nursery rootstock plants in Italy

Summary. Young grapevine plants with decline and wood necrosis symptoms were collected from vineyards and nurseries in the Apulia and Molise regions, Italy, from 2013 to 2015. Isolations of fungi were prepared from 45 diseased grapevine plants, and the cultures were identified. Several species commonly associated with Petri disease, Botryosphaeria dieback, and black foot disease were isolated. A detailed study was carried out, and 182 isolates resembling Cylindrocarpon-like asexual forms were identified through morphological characterisation and DNA analysis of internal transcribed spacer regions 1 and 2 of the rRNA gene and the partial β-tubulin gene. Dactylonectria torrensensis and Ilyonectria liriodendri were identified based on morphological features and the partial histone 3 gene, so these fungi can be defined as the causal agents of black foot on grapevine for the first time in Italy. Thelonectria blackeriella is also described as a new species, through morphological characterisation and multigenic analysis using sequence data for five loci (large subunit RNA, internal transcribed spacers, β-tubulin, actin, RNA polymerase II subunit 1). This new species was associated with black foot symptoms according to preliminary pathogenicity tests, with representative isolates of each of the three species. Pathogenicity tests showed that these species can cause black streaking in the wood of 1-year-old grapevine rootstock shoots. The identification of D. torresensis, I. liriodendri and T. blackeriella from young grapevine plants and rooted rootstock highlights the importance of black foot disease in Italy, which has previously been overlooked

Detection and quantification of black foot and crown and root rot pathogens in grapevine nursery soils in the Western Cape of South Africa

Black foot disease (BFD) and crown and root rot (CRR) are important soilborne diseases that affect young grapevines in nurseries and vineyards. A 3-year survey (2013–2015) of five open-field grapevine nurseries was conducted in the Western Cape Province of South Africa. The survey involved the isolation of BFD and CRR pathogens from grafted rootstocks (ten plants per nursery, per year) that were rooted in soil for 1 year. In 2013 and 2015, grapevines were sampled, while in 2014, sampling was focused on rotation crops and weeds (ten plants each). The rotation crops included white mustard, lupins, canola, triticale and forage radish. The weed species sampled included Johnson grass, ryegrass, winter grass, Cape marigold and corn spurry. Soil samples from ten sites per nursery were also collected in close proximity to the sampled plants, at depths of 0–30 cm and 30–60 cm (ten samples per depth). Isolations were made from the grapevines, rotation crops and weeds. Pathogen detection and quantification in the soil were determined using quantitative real-time polymerase chain reaction technology. The predominant BFD pathogens isolated from grapevines were Campylocarpon fasciculare, Ca. pseudofasciculare and Dactylonectria macrodidyma. The predominant CRR pathogens were Pythium irregulare and Phytopythium vexans. Dactylonectria macrodidyma, D. novozelandica, D. pauciseptata, Py. irregulare, Py. ultimum var. ultimum and Py. heterothallicum were isolated from triticale roots. Dactylonectria spp. were also isolated from corn spurry, while Py. irregulare and Py. ultimum var. ultimum were isolated from numerous weeds and rotation crops. Mean soil DNA concentrations of Ilyonectria and Dactylonectria were from 0.04 to 37.14 pg μL-1, and for Py. irregulare were between 0.01 and 3.77 pg μL-1. The Phytophthora mean soil DNA concentrations ranged from 0.01 to 33.48 pg μL-1. The qPCR protocols successfully detected and quantified BFD and CRR pathogens in grapevine nursery soil. This is the first report of D. pauciseptata and D. alcacerensis in South African grapevine nurseries

Evaluation of biocontrol agents for grapevine pruning wound protection against trunk pathogen infection.

Trunk diseases of grapevine are caused by numerous pathogens, including Eutypa lata, Phaeomoniella chlamydospora, and species of Botryosphaeriaceae (incl. Botryosphaeria and aggregate genera), Phomopsis and Phaeoacremonium. Since infections occur mainly through pruning wounds, that have been shown by previous research to stay susceptible for up to 16 weeks after pruning, long-term pruning wound protection is required for prevention of infection. This study evaluated several biocontrol agents against a range of trunk disease pathogens in dual plate laboratory trials to determine macroscopic and microscopic interactions. The biocontrol agents had a substantial effect on all the pathogens, with a wide range of macroscopic and microscopic interactions observed. The best performing biocontrol agents were tested in two field trials. Fresh pruning wounds were treated with benomyl, Trichoderma products (Biotricho®, Vinevax® and ECO 77®) and isolates (USPP-T1 and -T2, identified as T. atroviride) and Bacillus subtilis. Seven days after treatment the pruning wounds were inoculated by spraying with spore suspensions of Neofusicoccum australe, N. parvum, Diplodia seriata, Lasiodiplodia theobromae, Eutypa lata, Phaeomoniella chlamydospora or Phomopsis viticola. Eight months after inoculation, the treatments were evaluated by isolation onto potato dextrose agar. The efficacy of the biocontrol agents was in most cases similar or superior to that observed for benomyl. Isolate USPP-T1, in particular, was very effective, reducing incidence of Ph. viticola, E. lata, Pa. chlamydospora, N. australe, N. parvum, D. seriata and L. theobromae by 69, 76, 77, 78, 80, 85 and 92%, respectively. This is the first report of biological protection of grapevine pruning wounds against this group of grapevine trunk disease pathogens

Influence of water stress on Botryosphaeriaceae disease expression in grapevines

Several species in Botryosphaeriaceae have been associated with grapevine trunk diseases. To evaluate the effect of water stress on infection of grapevines by Botryosphaeriaceae spp., 1-year-old Shiraz/101-14 Mgt nursery grapevine plants were planted in plastic potting bags and placed outdoors under shade netting. Five weeks after planting, vines were pruned and the pruning wounds inoculated with spore suspensions of Neofusicoccum australe, Neofusicoccum parvum, Lasiodiplodia theobromae or Diplodia seriata. Control treatments consisted of applications of sterile water or a Trichoderma harzianum spore suspension. Stem inoculations were done by inserting a colonised or uncolonised agar plug into a wound made in each stem. Four different irrigation regimes were introduced 12 weeks after planting to simulate varying degrees of water stress. Measurements of stomatal conductance, photosynthetic rate and leaf spectrometry were made to monitor physiological stress. Eight months after inoculation, vines were uprooted and the root, shoot and plant mass of each vine determined. Lesions observed in the inoculated pruning wounds and stems were also measured. Vines subjected to the lowest irrigation regime were significantly smaller than optimally irrigated vines. Water stressed vines also had significantly lower photosynthetic rates and levels of stomatal conductance compared with vines receiving optimal irrigation, indicating that these plants experienced significantly higher levels of physiological stress. The mean lesion length was significantly longer in the pruning wounds and stems of plants subjected to the lowest irrigation regime, with lesion length declining linearly with increasing irrigation volume. These results clearly indicate that when a grapevine is exposed to water stress, colonisation and disease expression by Botryosphaeriaceae spp. are much more severe