Fungal pathogens associated with black foot of grapevine in China

Grapevine trunk diseases (GTDs) are the most destructive diseases in grape-growing regions worldwide. Black foot is one of the important GTDs affecting young vineyards and nurseries. This disease has not been reported in China. During 2017 and 2019, field surveys were carried out in the Guangxi, Hebei, Ningxia, Shanxi, and Xinjiang provinces of China. Incidence of plants with black foot symptoms was 0.1% to 1% in the surveyed vineyards. Plant samples with poorly developed shoots and canes, chlorotic leaves, and necrotic trunks or roots were collected from the five provinces. In total, 50 fungal isolates were obtained from symptomatic tissues. Based on morphological and multi-gene phylogenetic analyses, five species were identified as Cylindrocladiella lageniformis, Dactylonectria torresensis, D. macrodidyma, D. alcacerensis and Neonectria sp.1. Pathogenicity was assessed using young, healthy detached green shoots of grapevine ‘Summer Black’ and potted 3-month-old ‘Summer Black’ cuttings. Inoculated detached shoots developed necroses after 7 d, and inoculated cuttings after 80 d. Fungi were re-isolated from necrotic lesions. Among the five species, D. macrodidyma was the most aggressive. This is the first report of C. lageniformis, D. torresensis, D. macrodidyma, D. alcacerensis, and Neonectria sp. 1 associated with black foot in China. This study has enhanced knowledge of the fungi associated with black foot in China, and will assist development of control measures for this disease.

Evaluation of Sown Cover Crops and Spontaneous Weed Flora as a Potential Reservoir of Black-Foot Pathogens in Organic Viticulture

(1) Background. An extensive survey of grapevine-sown cover crops and spontaneous weed flora was conducted from 2019 to 2020 in organic vineyards in six European countries (France, Italy, Romania, Slovenia, Spain, Switzerland). Our main objective was to detect and identify the presence of Cylindrocarpon-like asexual morphs species associated with black-foot disease on their roots. (2) Methods. Fungal isolations from root fragments were performed on culture media. Cylindrocarpon-like asexual morph species were identified by analyzing the DNA sequence data of the histone H3 (his3) gene region. In all, 685 plants belonging to different botanical families and genera were analyzed. Cylindrocarpon-like asexual morphs were recovered from 68 plants (9.9% of the total) and approximately 0.97% of the plated root fragments. (3) Results. Three fungal species (Dactylonectria alcacerensis, Dactylonectria torresensis, Ilyonectria robusta) were identified. Dactylonectria torresensis was the most frequent, and was isolated from many cover crop species in all six countries. A principal component analysis with the vineyard variables showed that seasonal temperatures and organic matter soil content correlated positively with Cylindrocarpon-like asexual morphs incidence. (4) Conclusions. The presence of Cylindrocarpon-like asexual morphs on roots of cover crops suggests that they can potentially act as alternative hosts for long-term survival or to increase inoculum levels in vineyard soils.

Multi-Locus Phylogeny and Taxonomy of the Fungal Complex Associated With Rusty Root Rot of Panax ginseng in China

Panax ginseng rusty root rot caused by the Ilyonectria species complex is a devastating disease, and it is one of the main factors contributing to the difficulty in continual cropping. Rusty root rot occurs in all ginseng fields, but little is known about the taxonomy of the fungal pathogen complex, especially Ilyonectria and Ilyonectria-like species. Rusty root rot samples were collected from commercial ginseng cultivation areas of China, and the pathogens were isolated and purified as single spores. Based on the combination analysis of multiple loci (rDNA-ITS, TUB, HIS3, TEF, ACT, LSU, RPB1, RPB2, and SSU) and morphological characteristics, the pathogens causing ginseng rusty root rot were determined. Fungal isolates were obtained from infected roots in 56 locations within main cultivation areas in China. A total of 766 strains were identified as Ilyonectria, Ilyonectria-like and Rhexocercosporidium species, including I. robusta (55.0%), I. communis (21.7%), I. mors-panacis (10.9%), I. pseudodestructans (2.0%), I. changbaiensis (1.3%), I. qitaiheensis (1.3%), Neonectria obtusispora (2.0%), Dactylonectria torresensis (0.5%), D. sp. (0.5%), and R. panacis (1.5%), and four novel species, Thelonectria ginsengicola (1.0%), T. jixiensis (1.0%), T. mulanensis (0.8%) and T. fusongensis (0.5%), with a total of 14 species. As the pathogen present in the highest proportion, I. robusta was the most prevalent and damaging species, unlike the pathogens reported previously. All of the examined strains were proven to cause ginseng rusty root rot. Our results indicate that the taxonomy of the fungal complex associated with ginseng rusty root rot includes Ilyonectria, Ilyonectria-like genera (Dactylonectria, Neonectria, and Thelonectria) and Rhexocercosporidium.

Occurrence of Dactylonectria torresensis Causing Root Rot on Astragalus membranaceus in northeastern China

Astragalus membranaceus Bunge (Fabaceae) is a perennial medicinal herb widely cultivated in China. In June 2018, root rot was observed on two-year-old A. membranaceus plants in Chaoyangshan town (northeastern China). In a 40-ha field, over 40% of the plants exhibited root rot and the infected area ranged from 10 to 70% of the roots. The roots first exhibited circular or irregular brown, sunken and necrotic lesions, and finally multiple lesions coalesced. The infected root surface was destroyed, showing rusty and dry rot (Fig. 1). Symptoms were concentrated in the main roots (Carlucci et al. 2017). The aboveground parts of infected plants did not initially show symptoms but gradually wilted; 7.6% of the plants died when root decay became severe. Infected roots were not used for processing and were not marketable. Ten infected roots were collected from May to October 2018 from the above location. The diseased root tissue was cut into 25 mm3 pieces, immersed in 1% NaOCl for 2 minutes, rinsed three times with sterile water and placed on water agar in Petri plates. After 15 days of incubation at 20°C, 11 single-spore isolates were obtained. Isolates HQ1 and HQ2 were randomly selected for morphological and molecular identification. Colonies grown for 10 days produced yellow, cottony to felty aerial mycelium on potato dextrose agar. Conidiophores originating laterally or terminally from the mycelium were solitary to loosely aggregated and unbranched or sparsely branched. Macroconidia predominated and were cylindrical, with a tendency to gradually widen towards the tip; 1- to 3-septate; and 20.2 to 31.0 × 3.0 to 6.7 µm (n=100). Microconidia had mostly 0¬- to 1-septate and 8.6 to 16.7 × 1.9 to 5.1 µm (n=100) (Fig. 1). Chlamydospores were rare, but occasional chlamydospore chains were observed. The isolates were tentatively identified as Dactylonectria torresensis (Cabral et al. 2012a). Further confirmation of the two isolates was conducted by DNA sequencing of the internal transcribed spacer (ITS, GenBank accession no. MN558983 and MN558984), β-tubulin (TUB, MN561692 and MN561693), histone 3 (HIS3, MN561694 and MN561695), and translation elongation factor (TEF, MN561696 and MN561697) genes (Cabral et al. 2012b). These sequences had 99 to 100% match with D. torresensis (JF735362 for ITS, JF735492 for TUB, JF735681 for HIS3 and JF735870 for TEF). Phylogenetic trees based on analyses of a concatenated alignment of all loci grouped these isolates into the D. torresensis clade (Fig. 2). The same two isolates were tested for pathogenicity. Healthy two-year-old plants were taken from the field, and their roots were disinfected with 75% alcohol for 3 minutes, rinsed with sterile water three times, immersed in a 1×105/ml spore suspension or sterile water (control) for 10 minutes, transferred to a tray filled with sterile sand and placed in a greenhouse (12 h photoperiod, 25°C). Twelve plants grown in three pots were used for each isolate, and the same number of plants were inoculated as a control. This experiment was repeated three times. After one month, inoculated plant roots showed the same symptoms as those observed in the field, while the controls remained symptomless and no pathogen was recovered. The same fungus was reisolated from all the infected plants and confirmed by sequencing all of the above genes. This is the first report of D. torresensis causing root rot in A. membranaceus in China. The occurrence of this disease poses a threat, and management strategies need to be developed.

Comparative Genomic Analysis of Dactylonectria torresensis Strains from Grapevine, Soil and Weed Highlights Potential Mechanisms in Pathogenicity and Endophytic Lifestyle

The soil-borne fungus Dactylonectria torresensis is the most common causal agent of black-foot disease in Europe. However, there is a lack of understanding on how this fungus can provoke plant symptoms. In this study, we sequenced, annotated and analyzed the genomes of three isolates of D. torresensis collected from asymptomatic vine, weed and soil. Sequenced genomes were further compared to those of 27 fungal species including root and aerial pathogens, white rot degraders, indoor biodeterioration agents, saprotrophs, dark septate endophytes and mycorrhiza. Strains of D. torresensis present genomes with between 64 and 65 Mbp and with up to 18,548 predicted genes for each strain. Average Nucleotide Identity (ANI) shows that strains are different according to genome contents. Clusters of orthologous groups were compared, and clusters of genes related to necroses were particularly detected in all strains of D. torresensis (necrosis inducing peptides and proteins, and ethylene inducing peptides) as well as several genes involved in resistance against fungicides frequently used in viticulture such as copper. Interestingly, an expanded high number of genes related to carbohydrate-active enzymes were detected in each Dactylonectria strain, especially those related to glycoside hydrolases that could be involved in penetration of plant tissues or pathogenicity. An increased number of candidate genes for CAZyme classes AA9 and AA3-1 supports the ability of strains to efficiently degrade plant material. High numbers of genes of D. torresensis related to secretome and small secreted proteins were further characterized. Moreover, the presence of several gene clusters such as fujikurin-like genes was detected and were normally found in Fusariumfujikuroi, that have been linked to fungal pathogenicity. The phenotypes of the three strains investigated showed further difference in light response. We found that Dactylonectria strains have an increased number of photoreceptor encoding genes and we showed sequence alterations. Altogether, the results highlight several gene clusters present in D. torresensis strains that could be linked to endophytic lifestyle, pathogenicity, plant maceration and degradation of plant tissues as well as adaptation to soil contaminated with metals and metalloids and light response.

Alternativas de control orgánico in vitro para Dactylonectria torresensis en la mora de castilla (Rubus glaucus) en Ecuador

Esta investigación evaluó, en laboratorio, la eficiencia de los fungicidas de origen orgánico frente a los fungicidas químicos de uso convencional para controlar el crecimiento del Dactylonectria torresensis, considerada como una de las fitopatologías de importancia económica que afecta a la mora de castilla (Rubus glaucus) en Ecuador. El objetivo del estudio se basó en la necesidad imperante de buscar alternativas naturales y amigables con el ambiente, que permitan obtener alimentos limpios para la población. Para tal fin, se evaluó la eficiencia de los fungicidas convencionales de síntesis química: Azoxystrobin, Benomil, Carbendazim, Difeconazol, Fosetyl Aluminio, Tachigaren, Propiconazol, Penconazol, Metalaxil y Thiabendazole, cada uno en dosis de 100, 10 y 1 ppm, frente a la eficiencia de fungicidas de síntesis orgánica: aceite de neem, extracto de mirtáceas, extracto de ajo, sulfato cúplico pentahidratado y extracto de tomillo, aplicados en dosis de 300, 200, y 100 ppm. Se comprobó que el extracto de mirtáceas controló en 100 % del crecimiento micelial del Dactylonectria torresensis, y mostró resultados similares a los obtenidos con Carbendazim y Azoxystrobin. Se concluye, por lo tanto, que si existiera una alternativa para el control del hongo, esta podría ser utilizada comercialmente, y de esta manera disminuir el uso y los efectos nocivos de los agroquímicos convencionales.

Dactylonectria and Ilyonectria Species Causing Black Foot Disease of Andean Blackberry (Rubus Glaucus Benth) in Ecuador

Andean blackberry (Rubus glaucus Benth) plants from the provinces of Tungurahua and Bolivar (Ecuador) started showing symptoms of black foot disease since 2010. Wilted plants were sampled in both provinces from 2014 to 2017, and fungal isolates were obtained from tissues surrounding necrotic lesions in the cortex of the roots and crown. Based on morphological characteristics and DNA sequencing of histone 3 and the translation elongation factor 1α gene, isolates were identified as one of seven species, Ilyonectria vredehoekensis, Ilyonectria robusta, Ilyonectria venezuelensis, Ilyonectria europaea, Dactylonectria torresensis, or Dactylonectria novozelandica. Pathogenicity tests with isolates from each species, excluding I. europaea and D. novozelandica whose isolates were lost due to contamination, confirmed that the four species tested can produce black foot disease symptoms in Andean blackberry. This is the first report of Dactylonectria and Ilyonectria species causing black foot disease of Andean blackberry.