First Report of Diaporthe ambigua associated with dead arm disease on grapevine in Chile

Grapevine (Vitis vinifera L.) is one of the most important fruit crops in Chile based on economic value. Phaeomoniella chlamydospora and Botryosphaeriaceae species have been reported as the major causal agents associated with dieback symptoms in Chile commercial vineyards (Díaz and Latorre 2014; Besoain, 2018; Larach et al. 2020). Recently Eutypa lata has been reported attacking Chilean vineyards with dieback symptoms (Lolas et al. 2020). In this study, two commercial cv. Cabernet Sauvignon vineyards, located in O'Higgins Region of Chile, showing dead cordons, dead spur with a grayish color, canker, and vascular necrosis were sampled in fall 2018, with a high incidence of symptoms was observed. Four symptomatic wood samples were analyzed from these vineyards. Pieces of wood (<1 cm2) were taken from the advance zone of the canker lesions, disinfected with 70% ethanol, rinsed in sterile distilled water, dried, and transferred to two media in Petri plates, potato dextrose agar acidified with 0.5 ml of 96% lactic acid (APDA) and malt extract agar, and incubated for at least seven days at 24°C in darkness. From mycelium obtained from monosporic culture, two isolates were selected and morphologically identified as Diaporthe sp. To induce sporulation, these two isolates were grown in APDA under near-ultraviolet light (λ = 320 nm) at room temperature. After 30 days, the development of pycnidia was observed. Both Diaporthe sp. isolate presented alpha-conidia ellipsoidal with an obtuse apex, biguttulate (n=30) of 6.7 µm ± 0.33 µm x 3.3 µm ± 0.32 µm. No Beta-conidia or perhitecia were observed. DNA was extracted from the monosporic mycelium. The ribosomal internal transcribed spacer (ITS), β-tubulin (BT) gene, and elongation factor (EF) gene were amplified using ITS4/ITS5, Bt2a/Bt2b, and EF1-728F/EF1-986R primer pairs, respectively. PCR products were sequenced and identified as Diaporthe ambigua Nitschke (PUCV2140 and PUCV2141), showing 100% sequence identity with ITS MH864620.1, 99.8% with BT MG281142.1, and 100% with EF KC343738.1 sequences from D. ambigua. Sequences were deposited in GenBank (ITS: accession numbers MW301136, MW301137; BT: MW323445, MW323446 and EF: MW308305, MW308306). Two pathogenicity tests were performed with strains PUCV2140 and PUCV2141 using 2-year-old V.vinifera cv. Cabernet Sauvignon. In each test, three plants were used per isolate, considering one plant as an experimental unit. In the first test, a 5 mm mycelial plug from a 6-day-old APDA culture was inoculated using an oblique cut made in the bark with a sterile scalpel and done at the middle of the trunk. In the second test, the trial was done under the same described conditions previously, but in this case, one-year-old semi-lignified shoots were inoculated between two internodes, using mycelial plugs, one shoot for each plant. Injured plants but treated with sterile APDA plugs were used as controls. Plants were placed in natural conditions, and after three months from inoculations, plants showed a cortical canker and brown vascular lesions. Non-inoculated plants remained asymptomatic. The lengths of the cankers were 22.0 ± 1.8 mm and 10.5 ± 0.6 mm, after inoculations of the trunk and cane, respectively. The vascular lesions were 37.0 ± 3.3 and 18.0 ± 2.0 mm, in trunk and cane inoculations, respectively. D. ambigua was re-isolated and reidentified morphologically from the inoculated symptomatic plants, confirming Koch’s postulate. Also, the plants inoculated on the trunk showed premature leaf drop. To our knowledge, this is the first report of D. ambigua associated with dieback affecting grapevines in Chile. Previous D. ambigua was reported causing fruit rots (Auger et al. 2013; Díaz et al. 2017) and cordon dieback in kiwifruit (Díaz and Latorre, 2018), and stem canker and dieback in blueberry (Elfar et al. 2013) in Chile. This study reports a new species of fungi for Chile associated with the dead arm in vineyards. D. ambigua is a pathogen in essential crops in our country. Therefore, it is important to study its prevalence in the future.

Novel Seimatosporium Species from Grapevine in Northern California and Their Interactions with Fungal Pathogens Involved in the Trunk-Disease Complex

Seimatosporium spp. and closely related “pestalotioid fungi” have been isolated from vineyards worldwide, but their ecological status in grapevine wood is unclear. To determine their involvement in the grapevine trunk-disease complex, we tested the pathogenicity of Californian isolates obtained from vines with general symptoms of Botryosphaeria, Eutypa, and Phomopsis diebacks. Multilocus phylogenetic analyses revealed three species: Seimatosporium vitis and two newly described and typified species, S. luteosporum sp. nov. and S. vitifusiforme sp. nov. Inoculations to woody stems of potted grapevines of both isolates of S. vitis and one isolate of S. vitifusiforme, but not S. luteosporum, were associated with significantly larger lesions than those of noninoculated controls. Coinoculations with trunk pathogens (Cryptovalsa ampelina, Diaporthe ambigua, Diatrypella verruciformis, Diplodia seriata, and Eutypa lata), coisolated from the same wood cankers in the field, brought about increased lesion lengths for S. vitifusiforme paired with D. seriata, and S. luteosporum paired with Diaporthe ambigua. In contrast, there were no differences in lesion lengths of S. vitis and Diatrypella verruciformis or S. vitis and E. lata, inoculated alone or together. Our findings suggest that Seimatosporium spp. are involved in the grapevine trunk-disease complex, and their virulence may depend on or affect that of trunk pathogens.

Identification and Characterization of Diaporthe ambigua, D. australafricana, D. novem, and D. rudis Causing a Postharvest Fruit Rot in Kiwifruit

Diaporthe spp. are important plant pathogens causing wood cankers, blight, dieback, and fruit rot in a wide range of hosts. During surveys conducted during the 2013 and 2014 seasons, a postharvest rot in Hayward kiwifruit (Actinidia deliciosa) was observed in Chile. In order to identify the species of Diaporthe associated with this fruit rot, symptomatic fruit were collected from seven kiwifruit packinghouses located between San Francisco de Mostazal and Curicó (central Chile). Twenty-four isolates of Diaporthe spp. were identified from infected fruit based on morphological and cultural characters and analyses of nucleotides sequences of three loci, including the internal transcribed spacer (ITS) region (ITS1-5.8S-ITS2), a partial sequences of the β-tubulin, and translation elongation factor 1-α genes. The Diaporthe spp. identified were Diaporthe ambigua, D. australafricana, D. novem, and D. rudis. Multilocus phylogenetic analysis revealed that Chilean isolates were grouped in separate clades with their correspondent ex-types species. All species of Diaporthe were pathogenic on wounded kiwifruit after 30 days at 0°C under normal and controlled-atmosphere (2% O2 and 5% CO2) storage and they were sensitive to benomyl, pyraclostrobin, and tebuconazole fungicides. D. ambigua isolates were the most virulent based on the lesion length measured in inoculated Hayward and Jintao kiwifruit. These findings confirm D. ambigua, D. australafricana, D. novem, and D. rudis as the causal agents of kiwifruit rot during cold storage in Chile. The specie D. actinidiae, a common of Diaporthe sp. found associated with kiwifruit rot, was not identified in the present study.

Diaporthe ambigua Associated with Post-Harvest Fruit Rot of Kiwifruit in Chile

Kiwifruit is an important, expanding commercial crop in Chile. Several fungi have been reported to be associated with post-harvest rots of kiwifruit worldwide (2). During 2011 and 2012, kiwifruit produced in the VI and VII regions of Chile, showing symptoms of inner rot, were investigated with the aim of identifying the disease agent. The affected fruits had brown pubescent skin at the stem end that became soft and lighter in color than the adjacent firm healthy tissues. A watery exudate and white to pale grayish mycelial mats frequently developed at the stem end of the fruit, causing a water-drop stain down the sides on the dry brown healthy skin. The underlying tissue accessed by peeling off the skin was usually water soaked, soft, and lighter green than the healthy tissue. A fermented sour odor occurred frequently on severely decayed fruits. After incubation at 25°C over 7 days on potato dextrose agar (PDA), white to grayish, pale aerial mycelial mats were recovered from fragments of symptomatic kiwifruit superficially disinfected with 95% ethanol. After 2 weeks, black, spherical pycnidia developed, bearing hyaline, ellipsoidal, biguttulate conidia (5.4 to 12.6 × 2.1 to 4.7 μm). After 3 weeks, abundant perithecia embedded in a distinct, black, elevated stroma developed. Perithecia were black, globose, 200 to 500 μm in diameter with necks sinuous, filiform, 550 to 840 × 50 to 95 μm. Clavate, sessile asci, 30.9 to 50.2 × 6.6 to 12.5 μm contained ascospores biseriate, hyaline, smooth, fusoid-ellipsoid, widest just above the septum, tapering towards both ends, medianly septate, constricted at the septum at maturity, with 1 to 2 guttules per cell ascospores, 5.9 to 9.7 × 2.5 to 4.3 μm. All colonies obtained from kiwifruit displayed the same morphological traits and were consistent with those of a Diaporthe sp. (1). The internal transcribed spacer (ITS) region was sequenced using ITS1/ITS4 primers (4). Analysis of ITS region of kiwifruit isolates Damb_12 and Damb_16 (GenBank Accession Nos. KC294545 and KC294544, respectively) revealed 100% nucleotide identity to Diaporthe ambigua (HM575420, HM575419, DQ286274, DQ286270, and DQ286271). Six millimeter plugs from fungal colonies growing on PDA at 25°C for 7 days were used to inoculate 15 healthy untreated, ripe ‘Hayward' kiwifruits. Control fruits were inoculated with agar plugs. Inoculated fruits were incubated at 25°C and 80% relative humidity. After 7 days, white to pale grayish mycelial mats developed only on the inoculated fruits, releasing a water drop stain. The underlying tissue was lighter green and water soaked. D. ambigua was reisolated only from the inoculated fruits. D. actinidiae has been previously reported on kiwifruit canes in Chile (3); however, to our knowledge, this is the first report on the occurrence of D. ambigua (Phomopsis ambigua) on kiwifruit in Chile. The fungal isolates (no. 1 to 21) have been deposited in the Laboratorio de Fitopatología Frutal y Molecular, Departamento de Sanidad Vegetal, Facultad de Ciencias Agronómicas de la Universidad de Chile. References: (1) J. C. Jansen van Rensburg et al. Studies in Mycol. 55:65, 2006. (2) L. Luongo et al. J. Plant Pathol. 93:205, 2011. (3) A. Palma and E. Piontelli. E. Bol. Micol. 15:79, 2000. (4) White et al. PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, 1990.

A novel RNA mycovirus in a hypovirulent isolate of the plant pathogen Diaporthe ambigua

Hypovirulent isolates of the fruit tree fungal pathogen Diaporthe ambigua have previously been shown to harbour a double-stranded (ds)RNA genetic element of about 4 kb. In this study, we established the complete cDNA sequence of this dsRNA, which represents a replicative form of a positive-strand RNA virus that we have named D. ambigua RNA virus (DaRV). The nucleotide sequence of the genome is 4113 bp and has a GC content of 53%. Two large ORFs are present in the same reading frame. They are most probably translated by readthrough of a UAG stop codon in the central part of the genome. The longest possible translation product (p125) has a predicted molecular mass of about 125 kDa. A significant homology can be found to the non-structural proteins of carmoviruses of the positive-strand RNA virus family Tombusviridae. These proteins also include the conserved RNA-dependent RNA polymerase (RDRP) domain. In contrast to the genome organization of these plant viruses, no ORF is present at the 3′ end of the DaRV genome that encodes a coat protein. Therefore, it is proposed that DaRV is not encapsidated but that it occurs as RNA–RDRP complexes and/or that it might be associated with cell membranes. Interestingly, six putative transmembrane helices are predicted in the N-terminal part of p56 (translation product of the first ORF, N-terminal part of p125), which might direct and anchor the viral complex to membranes. DaRV is a mycovirus with a unique genome organization and has a distant relationship to the plant virus family Tombusviridae.