Diversity of Botryosphaeriaceae and Diaporthe species associated with postharvest apple fruit decay in Serbia

Family Botryosphaeriaceae and the genus Diaporthe (fam. Diaporthaceae) represent diverse groups of plant pathogens, which include causal agents of leaf spot, shoot blight, branch and stem cankers, dieback, and pre and postharvest apple fruit decay. Apple fruit with the symptoms of light to dark brown decay were collected during and after harvest from 2016 to 2018. Thirty selected isolates, which pathogenicity was confirmed, were identified and characterized based on multilocus phylogeny and morphology. Five species from the family Botryosphaeriaceae and two from the genus Diaporthe (fam. Diaporthaceae) were discovered. The most commonly isolated was Diplodia seriata followed by Botryosphaeria dothidea. In this work, Diaporthe rudis is described as a new postharvest pathogen of apple fruit. Diplodia bulgarica, Diplodia sapinea, Neofusicoccum yunnanense, and Diaporthe eres are initially described as postharvest apple and D. sapinea as postharvest quince and medlar fruit pathogens in Serbia. As species of the family Botryosphaeriaceae and the genus Diaporthe are known to cause other diseases on their hosts, have an endophytic nature, and a wide host range, findings from this study imply that they may become a new challenge for successful fruit production.

Characterization, Phytopathogenicity and Host Range Studies of Neoscytalidium Dimidiatuml. (Botryosphaeraceae) Associated With Dieback of Lebbeck Trees (Albizia Lebbeck L. Benth) in Saudi Arabia

A survey in 2016 showed that more than 80% lebbeck trees inside the main campus of Qassim University were wilted and dead. Symptoms of dieback, root rot, stem cankers and decline were observed in the trees. The trunks exhibited black masses of spores which soon spread to other, healthy trees. A fungus, having arthroconidial and asexual synanamorph characteristics, and was identified as Neoscytalidiumdimidiatum KSA of the class Coelomycetes within the family Botryosphaeraceae and was subsequently isolated from the infected lebbeck trees. Six-year-old lebbeck seedlings were inoculated with the N. dimidiatum KSA isolate. Symptoms of chloroses followed by dropping leaves appeared four weeks after inoculation. The fungus re-isolated from the infected seedlings expressed the same morphological characteristics on the culture media as the N. dimidiatum KSA isolate. A host range study involving six different tree species were inoculated under growth chamber conditions using the identified isolate of the N. dimidiatum KSA fungus. Four weeks after the inoculation, three of these species exhibited wilting and died. To the best of our knowledge, this study is the first to report on N. dimidiatum in Saudi Arabia.

Eucalyptus tereticornis (forest red gum).

Eucalyptus tereticornis is a versatile, fast growing and widely planted tree species. It has been especially successful in areas with summer rainfall followed by a moderate to severe dry season, although fast-growing provenances adapted to a winter rainfall are also available from its extensive natural distribution. The species is marginally less drought tolerant than its close relative E. camaldulensis but appears to be less susceptible than this species to leaf pathogens and stem cankers when planted in areas of seasonally-high humidity. Provenances have been identified from the southern, temperate part of the species distribution that are moderately frost and salt tolerant. Eucalyptus tereticornis is proving a valuable partner in various hydrid combinations with other fast growing eucalypts, including E. grandis and E. urophylla. The wood is used for a wide range of purposes including fuel, charcoal, paper, poles, posts, mining timber, hardboard and particleboard while the tree is used for shelterbelts, shade and in apiculture.

First Report of Neoscytalidium dimidiatum Causing Stem Canker on Dragon fruit (Hylocereus spp.) in Puerto Rico

Dragon fruit or pitahaya (Hylocereus spp.) is a tropical fruit belonging to the Cactaceae. It is native to Central and South America and commercially grown in the United States in southern California, south Florida and Puerto Rico. During a disease survey from April to June 2020, stem canker was observed in greenhouses and commercial orchards located in Mayaguez and San Sebastian, Puerto Rico with an incidence of 80%. Diseased cladodes (stems) of 1 mm2 tissue sections of 23 pitahaya varieties (NOI-13, NOI-14, NOI-16, N97-15, N97-17, N97-18, N97-20, N97-22, American Beauty, Cosmic Charlie, Halley’s comet, Purple Haze, Alice, Bloody Mary, Dark Star, David Bowie, Delight, Makisupa, Red Jaina, Soul Kitchen, Vietnamese Jaina, Neitzel and Lisa) were disinfested with 70% ethanol, rinsed with double distilled water and plated on potato dextrose agar (PDA) amended with 60 mg/L streptomycin. Three isolates (17B-173-T3, 12C-118-T1 and 13B-131-T2) of Neoscytalidium dimidiatum (syn. N. hyalinum) were identified using taxonomic keys (Crous et al., 2006) and sequencing of the internal transcribed spacer (ITS) with primers ITS5 and ITS4 (White et al. 1990) and translation elongation factor 1 alpha (TEF1-α) with primers EF1-728F and EF1-986R (Carbone and Kohn, 1999). Sequences were compared using the BLASTn tool with N. dimidiatum deposited in NCBI GenBank. In PDA, colonies of N. dimidiatum were initially powdery white and turned grayish-black with age. Arthroconidia (n=50) were dark brown, disarticulating, truncate or cylindrical at the base, thick-walled with 0 to 1 septum, averaging 9.1 X 5.5um in length. GenBank accession numbers of N. dimidiatum DNA sequences were MT921260, MT921261 and MT921262 for ITS and MT920898, MT920899 and MT920900 for TEF1-α. Sequences were 99-100% identical with Ex-isotype CBS145.78 accession numbers KF531816 for ITS and KF531795 for TEF1-α. Pathogenicity tests were conducted on 12 healthy dragon fruit plants of 1.5 years old using three non-detached cladodes per plant. Cladodes were inoculated with 5mm mycelial plugs from 8-day-old pure cultures grown on PDA. Three healthy dragon fruit plants were used as controls and were inoculated with PDA plugs only. The experiment was repeated once. Twenty days after inoculations (DAI), isolates of N. dimidiatum caused stem canker on dragon fruit plants. For all isolates, sunken orange spots averaged 3 X 2 mm in length at 8 DAI. Necrotic blotches with chlorotic halos averaged 10 X 15 mm at 14 DAI; stem cankers with water-soaked tissue were observed at 20 DAI, and arthroconidia and black pycnidia on dry stem cankers at 30 DAI. Untreated controls had no symptoms of stem canker, and no fungi were isolated from tissue. Neoscytalidium dimidiatum has been reported to cause stem canker on Hylocereus spp. in China, Florida, Israel, Malaysia and Taiwan (Chuang et al. 2012; Lan et al., 2012; Ezra et al., 2013; Sanahuja et al., 2016). To our knowledge, this is the first report of N. dimidiatum causing stem canker on dragon fruit in Puerto Rico. References: 1. Carbone, I., and Kohn, L. 1999. Mycologia, 91:553. doi:10.2307/3761358 2. Chuang, M. F. et al. 2012. Plant Disease 96: 906. https://doi.org/10.1094/PDIS-08-11-0689-PDN. 3. Crous, P. W., et al. 2006. Stud. Mycol. 55:235. https://doi.org/10.3114/sim.55.1.235 4. Ezra et al. 2013. Plant Disease 97: 1513. https://doi.org/10.1094/PDIS-05-13-0535-PDN 5. Lan, G.B. et al. 2012. Plant Disease 96: 1702. https://doi.org/10.1094/PDIS-07-12-0632-PDN 6. Sanahuja et al. 2016. Plant Disease 100: 1499. https://doi.org/10.1094/PDIS-11-15-1319-PDN 7. White, T., Bruns, T., Lee, S., and Taylor, J. 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. Pages 315-322 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, CA.

Evaluation of pyraclostrobin as a component in seed and foliar fungicides for the management of blackleg (Leptosphaeria maculans) of canola (Brassica napus)

Infection by Leptosphaeria maculans (Desm.) Ces. & de Not., the causal agent of blackleg of canola (Brassica napus L.), can result in seedling death, foliar lesions, and the development of stem cankers on adult plants. Strobilurin fungicides such as pyraclostrobin are popular, as they are effective against a broad range of fungal pathogens, have novel target specificity, and translaminar activity within the plant. The seed treatment Prosper FX (carbathiin + trifloxystrobin + metalaxyl) and an experimental formulation BAS 720 F (pyraclostrobin + fluxapyroxad + metalaxyl) were evaluated for their efficacy in managing blackleg when applied alone or in combination with the foliar fungicides Priaxor (pyraclostrobin + fluxapyroxad) or Tilt 250 EC (propiconazole). Experiments were conducted with the blackleg-susceptible cultivar ‘Westar’ and the moderately resistant cultivar ‘Dekalb 73-15 RR’ under greenhouse and field conditions in 2012 and 2013. In one greenhouse trial, all fungicide treatments significantly reduced stem blackleg severity on ‘73-15’ and ‘Westar’ compared with the inoculated–untreated controls. During 3 of 4 site-years, the combination of the experimental seed treatment BAS 720 F and Priaxor significantly reduced stem disease severity relative to the inoculated–untreated control by 60%–77% for ‘73-15’ and by 50%–75% for ‘Westar’. This treatment also resulted in a significant yield increase relative to the inoculated–untreated control in ‘Westar’ in 2 site-years by 24% and 56%. The results of the study suggest a reduction in blackleg symptoms and improved yields when canola is treated with a combination of seed and foliar fungicides, especially when cropping a susceptible cultivar.

Phytopythium litorale: A Novel Killer Pathogen of Plane (Platanus orientalis) Causing Canker Stain and Root and Collar Rot

Decline symptoms associated with lethal stem and branch canker stain along with root and collar rots were observed on 5- to 7-year-old roadside oriental plane trees (Platanus orientalis) in Diyarbakır, Turkey. Above-ground symptoms included leaf necrosis, leaf curling, extensive bluish or blackish staining of shoots, branches, stem bark, and wood surfaces, as well as stem cankers and exfoliation of branch bark scales. A general decline of the trees was distinctly visible from a distance. A Phytophthora/Pythium-like oomycete species with globose to ovoid, often papillate and internally proliferating sporangia was consistently isolated from the fine and coarse roots and stained branch parts and shoots. The pathogen was identified as Phytopythium litorale based on several morphological features. Partial DNA sequences of three loci, including nuclear rDNA internal transcribed spacer (ITS) and the large ribosomal subunit (LSU), and mitochondrial cytochrome c oxidase subunit II (coxII) confirmed the morphological identification. All P. litorale isolates were homothallic, developing gametangia, ornamented oogonia with elongate to lobate antheridia. Pathogenicity of P. litorale was tested by inoculation on excised shoots and by root inoculation on seedlings. P. litorale produced large lesions and blights on shoots in just 5 days and killed 100% of the seedlings in a month. This paper presents the first confirmed report of P. litorale as an important pathogen on a plant species causing branch and stem cankers, and root and collar rot, in and on P. orientalis, resulting in a rapid decline of trees and suggesting a threat to plane.

Botryosphaeriaceae Fungi as Causal Agents of Dieback and Corky Bark in Rambutan and Longan

Fungi in the Botryosphaeriaceae family cause dieback, fruit rots, and stem cankers in many tropical fruit trees. To identify which species of Botryosphaeriaceae were present in tropical fruit in Puerto Rico and the symptoms they cause in rambutan and longan, a disease survey was conducted throughout the island from 2008 to 2016. Diseased organs of rambutan, longan, mango, and tangerine were collected and 39 isolates belonging to the Botryosphaeriaceae family were isolated and identified. Phylogenetic analysis of three nuclear genes identified nine species: six Lasiodiplodia spp. and three Neofusicoccum spp. All 39 isolates were inoculated on healthy 1-year-old rambutan and longan seedlings to confirm their pathogenicity. Dieback on both rambutan and longan was observed at 14 days after inoculation (DAI). Fourteen isolates from seven Botryosphaeriaceae species (Lasiodiplodia brasiliensis, L. hormozganensis, L. iraniensis, L. pseudotheobromae, L. theobromae, Neofusicoccum batangarum, and N. parvum) caused dieback in rambutan. Five of these pathogenic isolates were collected from rambutan, four from longan, two from mango, and three from tangerine. Ten isolates of four Lasiodiplodia spp. caused dieback in longan: L. hormozganensis, L. iraniensis, L. pseudotheobromae and L. theobromae,. Of these, three were collected from rambutan, three from longan, one from mango, and three from tangerine. Abundant development of pycnidia on branches, called corky bark, were observed on both rambutan and longan 60 DAI. Cross-inoculations showed that pathogenicity is wide in spectrum, indicating that different planting alternatives should be considered for better crop management.

Fungi Associated with Damage Observed on Branches of Juglans nigra in Indiana, Missouri, and Tennessee

Branch and stem cankers caused by Geosmithia morbida associated with mass attack by its primary insect vector (Pityophthorus juglandis) result in thousand cankers disease (TCD) on Juglans and Pterocarya species. Because other fungi and insects can cause visible damage to Juglans nigra, a baseline assessment was performed to document damage types present and to characterize fungi associated with each type. Two branches were collected from trees with visually healthy crowns in TCD-free locations (Indiana and Missouri) and two branches from trees with and without crown symptoms characteristic of TCD within the disease range in Tennessee. In most cases, one of the two branches was girdled at the base 3 to 4 months prior to harvest. Outer bark was peeled from branch subsamples, observed damage characterized, and isolation of fungi from each damage type attempted. Three known pathogens of J. nigra were obtained from different damage types: G. morbida, in Tennessee only; Botryosphaeria dothidea, in Indiana and Tennessee; and Fusarium solani (= members of F. solani species complex), in all three states. The latter two fungi may exacerbate branch dieback and mortality of TCD-affected trees. These results will be of value to plant health specialists monitoring J. nigra in the field and laboratory diagnosticians processing survey samples.

Phylogeny, Distribution, and Pathogenicity of Lasiodiplodia Species Associated With Cankers and Dieback Symptoms of Persian Lime in Mexico

Persian lime (Citrus latifolia Tan.) is an important and widely cultivated fruit crop in several regions of Mexico. In recent years, severe symptoms of gummosis, stem cankers, and dieback were detected in the Persian lime-producing region in the states of Veracruz and Puebla, Mexico. The aims of this study were to identify the species of Lasiodiplodia associated with these symptoms, determine the distribution of these species, and test their pathogenicity and virulence on Persian lime plants. In 2015, symptomatic samples were collected from 12 commercial Persian lime orchards, and 60 Lasiodiplodia isolates were obtained. Fungal identification of 32 representative isolates was performed using a phylogenetic analysis based on DNA sequence data of the internal transcribed spacer region and part of the translation elongation factor 1-α and β-tubulin genes. Sequence analyses were carried out using the Maximum Likelihood and Bayesian Inference methods. Six Lasiodiplodia species were identified as Lasiodiplodia pseudotheobromae, Lasiodiplodia theobromae, Lasiodiplodia brasiliense, Lasiodiplodia subglobosa, Lasiodiplodia citricola, and Lasiodiplodia iraniensis. All Lasiodiplodia species of this study are reported for the first time in association with Persian lime in Mexico and worldwide. L. pseudotheobromae (46.9% of isolates) was the most frequently isolated species followed by L. theobromae (28.1%) and L. brasiliense (12.5%). Pathogenicity on Persian lime young plants using a mycelial plug inoculation method showed that all identified Lasiodiplodia species were able to cause necrotic lesions and gummosis, but L. subglobosa, L. iraniensis, and L. pseudotheobromae were the most virulent.