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.     

黄瓜枯萎病菌毒力、营养体亲和性及ISSR分析

 本研究对来自哈尔滨、长春、沈阳、北京、西宁5个城市的70个尖孢镰刀菌黄瓜专化型菌株进行了毒力、营养体亲和性及ISSR分析。毒力测定结果显示黄瓜枯萎病菌在东农803品种上存在明显的毒力分化。在营养体亲和群的测定中有8个菌株没有产生nit突变体,2个菌株经测定为异核体自身不亲和性菌株,不能进行营养体亲和群的测定;其余60个菌株可分为5个营养体亲和群。利用筛选的7个引物对70个菌株进行了ISSR分子标记,聚类分析可将70个菌株分为3个类群,其中IGⅠ的41个菌株均来自东北三省,IGⅡ的21个菌株均来自北京,IGⅢ的8个菌株全部来自西宁。VCGs和ISSRs与菌株的地理来源及毒力存在一定的相关性。     

Restriction fragment length polymorphisms, races and vegetative compatibility groups within a worldwide collection of Fusarium oxysporum f.sp. gladioli

Isolates of Fusarium oxysporum f.sp. gladioli were collected from widely different geographic areas. These isolates were characterized by pathogenicity to two differential gladiolus cultivars, vegetative compatibility, and total genomic DNA restriction fragment length polymorphisms (RFLPs). RFLPs were used to estimate the genetic divergence and relationship among isolates of F. oxysporum. RFLPs were detected by Southern blot hybridization of total genomic DNA with a 3-4 kb DNA probe generated from total DNA off. oxysporum f.sp. dianthi. Cluster analysis allowed the division of pathogenic strains into three main RFLP groups, each group containing strains with similarity coefficients ranging from 78 to 100%. RFLP groups correlated with vegetative compatibility groups, not with races. Two single pathogenic isolates which could not be assigned to any of the three main vegetative compatibility groups also had distinctive RFLP patterns. Little genetic polymorphism was observed within vegetative compatibility groups, whereas the majority of RFLPs occurred between vegetative compatibility groups, suggesting a common ancestry for strains within a specific vegetative compatibility group and a polyphyletic origin for the present special form gladioli.     

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.     

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.     

Genetic variation,vegetative compatibility,and aggressiveness diversity of Diplodia bulgarica isolates from apple orchards in West Azarbaijan province of Iran

This study investigated inter-simple sequence repeat (ISSR), vegetative compatibility, and aggressiveness diversity in 101 isolates of Diplodia bulgarica recovered from apple trees displaying symptoms of canker and decline in West Azarbaijan province of Iran. Marker analyses revealed high within population diversity, low genetic differentiation, high gene flow, and sharing of multilocus genotypes (MLGs) among geographic populations. Moreover, clustering and multivariate analyses identified two highly differentiated genetic clusters with limited admixture between them. These findings may suggest that the pathogen has been introduced from two genetically divergent sources and has been moved within the region through infected materials. The large number of MLGs, low clonal fraction, and absence of a widely distributed dominant genotype may explain the occurrence of recombination in this pathogen. However, significant linkage disequilibrium in the populations and limited admixture between genetic clusters may indicate the rare occurrence of recombination in D. bulgarica populations in West Azarbaijan, and that the pathogen has not been in the province long enough to reach equilibrium. Vegetative compatibility analyses revealed the occurrence of anastomosis between nonself pairings and high vegetative compatibility group diversity within populations. All studied MLGs produced necrotic lesions on detached shoots of Red Delicious apple but differed in their aggressiveness levels. Our results provide new insights into genetic and phenotypic variation of D. bulgarica that can assist in developing management strategies. Our findings also highlight the vital need for quarantine measures and the production of healthy plant materials to prevent the introduction and spread of the pathogen in Iran.     

Characterisation ofFusarium oxysporum isolated from carnation in Australia based on pathogenicity,vegetative compatibility and random amplified polymorphic DNA (RAPD) assay

Isolates ofF. oxysporum collected from symptomless carnation cuttings from Australian carnation growers properties, together with isolates from national collections, were screened for pathogenicity and grouped according to vegetative compatibility and random amplified polymorphic DNA (RAPD) patterns. The collection of 82 Australian isolates sorted into 23 different vegetative compatibility groups (VCGs). Of 69 isolates tested for pathogenicity, 24 were pathogenic to carnations, while the remaining 45 were non-pathogenic. All pathogenic isolates were within two VCGs, one of which was also compatible with an isolate obtained from an international culture collection, and which is known to represent VCG 0021 and race 2. Race status of the two pathogenic VCGs remains unknown. The RAPD assay revealed distinct DNA banding patterns which could distinguish pathogenic from non-pathogenic isolates as well as differentiate between isolates from the two pathogenic VCGs.     

Physiological races and vegetative compatibility groups within Fusarium oxysporum f.sp. gladioli

The pathogenicity and vegetative compatibility of mainly Dutch isolates ofFusarium oxysporum collected from diseased gladioli and other Iridaceae were investigated. Based on their pathogenicity to two differential gladiolus cultivars, the isolates could tentatively be divided into two races. All self-compatible isolates ofFusarium oxysporum f.sp.gladioli belonged to one of three distinct vegetative compatibility groups, VCG 0340, 0341 or 0342, and were incompatible with isolates that were not pathogenic to gladiolus. Isolates of one of the two races were restricted to one VCG while isolates of the other race were present in all three VCGs.     

Diversity and pathogenicity of Fusarium graminearum species complex from maize stalk and ear rot strains in northeast China

The Fusarium graminearum species complex (FGSC) is an important group of pathogens distributed in maize‐producing areas worldwide. This study investigated the genetic diversity and pathogenicity of 40 FGSC isolates obtained from stalk rot and ear rot samples collected from 42 locations in northeastern China during 2013 and 2014. A phylogenetic tree of translation elongation factor (EF‐la) sequences designated the 40 isolates as F. graminearum sensu stricto (67.5%) and F. boothii (32.5%). By using inter‐simple sequence repeat analysis (ISSR), it was shown that the isolates were divided into two clades, which corresponded to the species identity of the isolates. However, the isolates from the two different diseases could not be distinguished in pathogenicity. The disease severity index of seedlings inoculated with stalk isolates was slightly higher than that of seedlings inoculated with isolates from infected ears, whereas the pathogenicity of the stalk and ear isolates were identical.     

Characterization of <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f. sp. <Emphasis Type="Italic">melongenae</Emphasis> isolates from eggplant in Turkey by pathogenicity,VCG and RAPD analysis

Fusarium wilt is an economically important fungal disease of common eggplant (Solanum melongena) cultivated in the eastern Mediterranean region of Turkey. Seventy-four isolates of Fusarium oxysporum isolated from diseased eggplant displaying typical Fusarium wilt symptoms were screened for pathogenicity on the highly susceptible cv. ‘Pala’. All the isolates tested were pathogenic to eggplant and designated as Fusarium oxysporum f. sp. melongenae (Fomg). Genetic diversity among a core set of 20 Fomg isolates that were selected based upon geographic locations, were characterized by using pathogenicity, vegetative compatibility grouping (VCG), and random amplified polymorphic DNA (RAPD) analysis. The area under the disease progress curve (AUDPC) was calculated for each Fomg isolate until 21 days after inoculation (DAI). The most virulent isolate was identified as Fomg10 based on AUDPC, disease severity and vascular discoloration measurements at 21 DAI. At this date, a good correlation was observed between disease severity and AUDPC values for all isolates (r = 0.73). UPGMA (unweighted pair group method with arithmetic average) cluster analysis of RAPD data using Dice’s coefficient of similarity differentiated all the Fomg isolates tested, and indicated considerable genetic variation among Fomg isolates, but isolates from the same geographic region were grouped together. There was no direct correlation between clustering in the RAPD dendrogram and pathogenicity testing of Fomg isolates. Twenty isolates of Fomg were assigned to VCG 0320.     

Characterization of Cytospora isolates from wood cankers of declining grapevine in North America,with the descriptions of two new Cytospora species

Cytospora species are ubiquitous pathogens of numerous woody plants, causing dieback and wood cankers in agronomic crops, timber trees and wildland trees (e.g. Prunus, Eucalyptus and Salix, respectively). Cytospora chrysosperma, C. cincta and C. leucostoma have been reported from grapevines in Iran showing symptoms of one or more recognized trunk diseases (esca, botryosphaeria‐, eutypa‐ and phomopsis diebacks); however, only C. chrysosperma was shown to be pathogenic to grapevine. To understand the potential role of Cytospora species in the grapevine trunk‐disease complex, 21 Cytospora isolates were examined that were recovered from dieback and wood cankers of Vitis vinifera and Vitis interspecific hybrids in seven northeastern U.S. states and two Canadian provinces. Phylogenetic analyses of ITS and translation elongation factor 1‐α identified two novel species: Cytospora vinacea sp. nov. and Cytospora viticola sp. nov. Differences in culture morphology and conidial dimensions also distinguished the species. When inoculated to the woody stems of potted V. vinifera ‘Thompson Seedless’ in the greenhouse, both species were pathogenic, based on development of wood lesions and fulfilment of Koch's postulates. Cytospora viticola was the most virulent based on lesion length at 12 months post‐inoculation. As cytospora canker shares some of the same general dieback‐type symptoms as botryosphaeria‐, eutypa‐ and phomopsis diebacks, it may be considered part of the grapevine trunk‐disease complex in eastern North America.     

Vegetative-compatibility groups in Verticillium dahliae from Israel

Nitrate-nonutilizing (nit) mutants were used to determine vegetative compatibility among 34 isolates of Verticillium dahliae from cotton, potato, olive, eggplant, chrysanthemum and tomato from 12 sites in Israel. Based on the formation of complementary heterokaryons, 33 isolates were assigned to two vegetative- compatibility groups (VCGs): one VCG contained 15 isolates from cotton, eggplant, chrysanthemum and olive; and the other VCG contained 18 isolates from potato, olive and cotton. The status of an additional isolate from tomato, which was compatible with both VCGs, remained unclear. In a limited pathogenicity test with 10 isolates, two (from tomato and eggplant) were pathogenic on tomato, eggplant and cotton; most isolates from cotton were pathogenic on cotton and eggplant only; and one from cotton was non-pathogenic. Fewer isolates were pathogenic on tomato than on cotton or eggplant. The diversity of vegetative compatibility found in our V. dahliae collection is comparable to that found in studies of American populations.     

Genetic diversity and pathogenicity of Monilinia polystroma – the new pathogen of cherries

Brown rot caused by fungi belonging to the genus Monilinia is one of the major limiting factors of sour and sweet cherry production. Up to now, three species, M. fructigena, M. laxa and M. fructicola, have been identified as causal agents of brown rot on cherries worldwide. From 2010 to 2013, during the monitoring of cherry orchards in different areas of Poland, a fourth species, M. polystroma, was isolated from brown rot symptoms on sour and sweet cherry fruits. To the best of the authors’ knowledge, this is the first time M. polystroma has been reported as the causal agent of brown rot on cherries. The genetic diversity of M. polystroma isolates from cherries and other hosts was analysed using PCR MP, ISSR and RAPD techniques and showed its clear distinctness from other Monilinia spp. tested. The cluster analysis of fingerprinting data revealed a high similarity of M. polystroma isolates from Poland and their close relationship with the reference strain from Japan, indicating that this species is a recently introduced pathogen. The highest genetic distance between the examined isolates and the highest number of different genotypes was observed in an ISSR assay. Detailed genetic diversity characteristics revealed that M. polystroma isolates from cherries did not create a distinct group but were intermingled with M. polystroma isolates from other hosts. The results of the pathogenicity test conducted on different fruit species indicated a lack of host specificity for M. polystroma isolates.     

Vegetative compatibility grouping in Botrytis cinerea using sulphate non-utilizing mutants

Twenty-one strains of Botrytis cinerea isolated from six plant species on ten sites throughout Israel, as well as a strain from France, were tested for vegetative and mycelial incompatibility, pathogenicity, resistance to the fungicides carbendazim and iprodione, and colony morphology. Selenate-resistant mutants were isolated from the strains as spontaneous, fast-growing sectors arising from restricted colonies on medium amended with sodium selenate with a mean frequency of 0.04 sectors/colony; 81% of the sectors were sulphate non-utilizing (sul) mutants. One hundred and four sul mutants were divided into two complementary groups: resistant (66 mutants) and sensitive to chromate. Based on compatibility reactions between chromate-resistant and chromate-sensitive sul mutants, 12 strains were compatible only with themselves and were each classified as belonging to different vegetative compatibility groups (VCGs). Nine strains were each compatible with one to three other strains, and were assembled into three multi-member VCGs. Mycelial incompatibility between wild-type strains (barrage), in the form of a zone of dark pigmentation or sparse mycelium with or without dark pigmentation of the agar along the line of confrontation, was observed for 70% of the inter-strain pairings. There was no correspondence in compatibility between strains revealed by two approaches: strains in different VCGs did not necessarily produce a barrage. However, self-compatibility was observed both as heterokaryon formation between complementary sul mutants and as an absence of barrages between mycelia of wild-type strains; wild-type strains belonging to the same VCG did not exhibit strong barrages, although weak antagonistic reactions were observed. Strains in two multi-member VCGs showed the same patterns of resistance to carbendazim and iprodione; the third multi-member VCG contained isolates with different patterns of resistance. Four morphological types were revealed among wild-type strains: conidial (five strains), sclerotial (six strains), intermediate (ten strains), and mycelial (one strain). On bean leaves, conidial strains were more aggressive than sclerotial strains.     

Molecular diversity of binucleate <Emphasis Type="Italic">Rhizoctonia</Emphasis> AG-A in China

AG-A belongs to the binucleate Rhizoctonia (BNR) anastomosis group (AG) of the Ceratobasidium teleomorph, which parasitizes the roots of many plant species. Ninety nine isolate species of AG-A were obtained from Tibet, Sichuan, and Yunnan Province in China. All isolates were divided into three types based on their cultural characteristics. Type I: abundant aerial mycelia, dense hyphae, loose sclerotia; Type II: abundant aerial mycelia, no sclerotia. Type III: sparse aerial mycelium and no sclerotia. All of the isolates infected the seedlings of Chinese mustard and Chinese cabbage, causing the formation of lesions on the stem and a brown discoloration of the roots. Sequence analysis of the 5.8S rDNA-ITS showed a similarity of 98–100% among the isolates. Inter Simple Sequence Repeat (ISSR) was used to detect genetic variation in binucleate Rhizoctonia spp. Forty two AG-A isolates were amplified using 15 random primers. From a total of 164 bands, 144 bands (87.8%) were polymorphic in the 42 tested isolates. A dendrogram showing genetic relationships between the isolates was constructed using unweighted pair-group averages based on genetic distances. According to the dendrogram, the 42 tested isolates could be aligned into three clusters with a genetic similarity coefficient of 0.29, the first clusters including 27 isolates with III of culture characteristics on PDA; the second clusters included eight isolates with I of cultural characteristics on PDA; the third cluster included seven isolates with II of cultural characteristics on PDA. The results of ISSR analysis showed an association between the hosts of these isolates. Our results showed that ISSR analysis can reveal more molecular variation among isolates of AG-A than sequence analysis using the 5.8S rDNA-ITS.     

Vegetative compatibility of Fusarium oxysporum f.sp. dianthi from carnation in Israel

Auxotrophic mutants were used to determine vegetative relatedness among isolates of Fusarium oxysporum f.sp. dianthi (F.o.d.) , the vascular wilt pathogen of carnation. At the first stage, different nitrate-non-utilizing (nit) mutants were produced from 11 isolates of F.o.d. collected in Israel. Complementation (heterokaryon) tests showed that all the isolates belonged to a single vegetative compatibility group (VCG), and two mutants were chosen as its testers. Additional isolates of Fusarium from carnation, collected during 1986-88, were analysed for pathogenicity and vegetative compatibility with the testers. A total of 170 Fusarium isolates, obtained from 42 cultivars at 40 sites, were tested. All the nit mutants of all the 132 pathogenic isolates formed heterokaryons with the testers, indicating that they belonged to the same VCG. None of the 38 non-pathogenic isolates was vegetatively compatible with the testers. The nit mutants retained pathogenicity to carnation. The F.o.d. testers were not compatible with testers of five other formae speciales of F. oxysporum. Thus, F.o.d. appears to constitute a distinct genetic population within the F. oxysporum complex.     

福建省丙环唑不同敏感性玉米小斑病菌的遗传多样性和致病性

 利用菌丝生长法、ISSR分子标记和分生孢子悬浮液喷雾接种法,探讨了福建省玉米小斑病菌对丙环唑的敏感性以及不同敏感性病菌群体的遗传多样性和致病性。敏感性测定结果表明,福建省玉米小斑病菌对丙环唑产生了抗药性,抗药性菌株的抗性倍数达到2.1~9.4倍。筛选获得的10条ISSR引物对55个菌株共检测出153个位点,其中多态性位点百分比高达93.46%。在敏感型、中间型和抗药型群体中多态性位点百分比分别为77.12%、69.93%和81.70%,在抗药型群体中,等位基因观测值、等位基因有效值、Nei’s遗传多样性指数和Shannon’s信息指数均高于敏感型群体,表明玉米小斑病菌抗药型群体的遗传多样性最丰富。聚类分析结果表明,病菌群体遗传多样性与抗药性水平和地理来源均有较高的相关性。致病性测定表明,丙环唑不同敏感性病菌群体对11个鲜食玉米品种均具有较强的致病性,但是,在9个玉米品种上,敏感型群体中强致病力菌株出现频率明显低于抗药型群体。研究结果为深入研究玉米小斑病菌群体遗传结构及其田间抗药性监测提供了理论基础。     

Identity and variability of Pythium species associated with yield decline in aerobic rice cultivation in the Philippines

The cultivation of aerobic rice in the tropics enables farmers to save water without lowering productivity. Unfortunately, this system suffers from declining yields due to a disease complex involving nematodes, pathogenic Pythium spp. and nutrient deficiencies. Assessing the impact of each underlying factor can contribute to efficient disease control measures. This study therefore investigated pathogenic and genotypic variability among Pythium species from affected aerobic rice fields in the Philippines using pathogenicity assays and sequence information from the internal transcribed spacer (ITS) region and β‐tubulin gene. Three closely related Pythium spp., P. arrhenomanes, P. graminicola and P. inflatum, were recovered from affected aerobic rice fields. All P. arrhenomanes isolates reduced rice seedling growth, whereas only a few P. graminicola isolates and no P. inflatum isolates were pathogenic, indicating that P. arrhenomanes is probably the most important species affecting rice. Both P. arrhenomanes and P. graminicola isolates showed little genetic variation, despite the observed pathogenic variation within P. graminicola. Intraspecific variation was higher among P. inflatum isolates, but again no correlation was observed with phenotype. When screening P. arrhenomanes isolates from other hosts such as sugarcane, maize and several grasses, a link between pathogenic and genetic variability was detected. However, rice and maize isolates seemed to lack host specificity, and therefore crop rotation with maize might be a risky strategy to manage yield decline in Philippine aerobic rice fields.     

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.     

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.