A Selective Medium for the Recovery and Enumeration of Fomitopsis meliae, Causing Lemon Canker and Wood Rot

A selective medium (FMS medium) was developed for the isolation and quantification of Fomitopsis meliae, the causal agent of lemon canker and brown wood rot, from plants, soil, and air. The optimal concentration and combination of fungicides and antibiotics was evaluated to determine the most selective condition for growing F. meliae. The resultant composition of the medium (FMS) per litre (pH 3.5) was: 16 mg thiophanate-methyl, 8 mg dichloran, 5 mg 2-phenylphenol, 100 mg fluopyram, 0.5 mg fludioxonil, 100 mg chloramphenicol, 100 mg streptomycin, 15 g malt extract, 2.5 g mycological peptone, and 15 g agar. The fungus was successfully isolated and enumerated from air, soil and plant tissues using FMS medium. Furthermore, FMS medium almost completely inhibited the growth of other plant pathogenic fungi, soil and air saprophytes. This selectivity was high enough to estimate spore inoculum of F. meliae in an air sample or as a spore trapping device in commercial lemon orchards. FMS medium will be useful for studying epidemiology and management of F. meliae.

Assessing the Use of DNA Detection Platforms Combined With Passive Wind-Powered Spore Traps for Early Surveillance of Potato and Tomato Late Blight in Canada

Quantitative PCR (qPCR), loop-mediated amplification (LAMP), and lateral flow strip-based recombinase polymerase amplification (RPA-LFS) assays were assessed for early detection of Phytophthora infestans, the global causal agent of potato and tomato late blight, on passive wind-powered spore traps known as Spornados. Spore traps were deployed in potato and tomato fields during the 2018, 2019, and 2020 growing seasons in the provinces of Alberta, British Columbia, Manitoba, Prince Edward Island, and Ontario. All assays used DNA extracts from Spornado cassette membranes targeting the P. infestans nuclear ribosomal internal transcribed spacer. A total of 1,003 Spornado samples were qPCR tested, yielding 115 positive samples for P. infestans spores. In further assessment of these samples, LAMP detected P. infestans in 108 (93.9%) of 115 qPCR positive samples, and RPA-LFS detected it in 103 (89.6%). None of the assays showed cross-reaction with other Phytophthora species or pathogenic fungi known to infect potato and tomato. The qPCR detected ≤1 fg of P. infestans DNA, and LAMP and RPA-LFS amplified 10 fg in as little as 10 min. All assays detected P. infestans before the first report of late blight symptoms in commercial potato or tomato fields within each region or province. The combination of Spornado passive samplers with qPCR, LAMP, or RPA-LFS proved a valuable spore trapping system for early surveillance of late blight in potato and tomato. Both LAMP and RPA-LFS showed potential as alternative approaches to qPCR for in-field monitoring of P. infestans.

A SYBR Green qPCR Method for Detecting and Quantifying Spores of Colletotrichum acutatum and C. gloeosporioides Species Complexes Causing Ripe Rot of Grape

Four species belonging to Colletotrichum acutatum and Colletotrichum gloeosporioides species complexes, including C. aenigma, C. fioriniae, C. fructicola, and C. nymphaeae, were identified from ripe rot symptomatic fruit from Mid-Atlantic vineyards. A quantitative real-time PCR (qPCR) SYBR Green method was developed to detect and quantify conidia of the two Colletotrichum complexes to better understand the distribution and the extent of Colletotrichum inoculum. Primers were designed to specifically target the β-tubulin gene of the C. acutatum complex or the C. gloeosporioides complex and were found to be specific to the desired complex, not amplifying DNA of other prevalent fungi from vineyards. The sensitivity of the primers was also assessed, and the C. acutatum complex-specific and C. gloeosporioides complex-specific primers were able to quantify as little as 50 C. fioriniae conidia and 100 C. aenigma conidia, respectively. Therefore, this would be a cost-effective, specific, and sensitive detection and quantification method for these two destructive species complexes in vineyards and many other crops through spore trapping applications.

Comparison of qPCR and Metabarcoding Methods as Tools for the Detection of Airborne Inoculum of Forest Fungal Pathogens

Forest diseases caused by invasive fungal pathogens are becoming more common, sometimes with dramatic consequences to forest ecosystems. The development of early detection systems is necessary for efficient surveillance and to mitigate the impact of invasive pathogens. Windborne spores are an important pathway for introduction of fungal pathogens into new areas; the design of spore trapping devices adapted to forests, capable of collecting different types of spores, and aligned with development of efficient molecular methods for detection of the pathogen, should help forest managers anticipate new disease outbreaks. Two types of Rotorod samplers were evaluated for the collection of airborne inoculum of forest fungal pathogens with a range of spore sizes in five forest types. Detection was by specific quantitative PCR (qPCR) and by high-throughput sequencing (HTS) of amplified internal transcribed spacer sequences using a new bioinformatic pipeline, FungiSearch, developed for diagnostic purposes. Validation of the pipeline was conducted on mock communities of 10 fungal species belonging to different taxa. Although the sensitivity of the new HTS pipeline was lower than the specific qPCR, it was able to detect a wide variety of fungal pathogens. FungiSearch is easy to use, and the reference database is updatable, making the tool suitable for rapid identification of new pathogens. This new approach combining spore trapping and HTS detection is promising as a diagnostic tool for invasive fungal pathogens.

New species of Phaeomoniellales from a German vineyard and their potential threat to grapevine (Vitis vinifera) health

Recently, the order Phaeomoniellales was established that includes fungi closely related to Phaeomoniella chlamydospora, a phytopathogen assumed to be the main causal agent of the two most destructive grapevine trunk diseases, Petri disease and esca. Other species of this order are reported as pathogens of other economically important crops, like olive, peach, apricot, cherry, plum, rambutan, lichee or langsat. However, they are rarely isolated and hence, little is known about their ecological traits and pathogenicity. During a 1-yr period of spore trapping in a German vineyard divided in minimally and intensively pruned grapevines, 23 fungal strains of the Phaeomoniellales were collected. Based on morphological and molecular (ITS, LSU and tub2) analyses the isolated strains were assigned to eight different species. Two species were identified as P. chlamydospora and Neophaeomoniella zymoides, respectively. The remaining six species displayed morphological and molecular differences to known species of the Phaeomoniellales and are newly described, namely Aequabiliella palatina, Minutiella simplex, Moristroma germanicum, Mo. palatinum, Neophaeomoniella constricta and N. ossiformis. A pathogenicity test conducted in the greenhouse revealed that except for P. chlamydospora, none of the species of the Phaeomoniellales isolated from spore traps is able to induce lesions in grapevine wood.

Inoculum Dynamics and Infection of Citrus Fruit by Phyllosticta citricarpa

Citrus black spot, caused by Phyllosticta citricarpa, is characterized by fruit blemishes and premature fruit drop, resulting in significant economic losses in summer rainfall areas. The pathogen forms both conidia and ascospores during its life cycle. However, the occurrence of these spores and their contributions to infection of fruit in field conditions are not well understood. Our research using direct leaf litter monitoring and volumetric spore trapping in Queensland orchards revealed that pseudothecia and ascospores in leaf litter as well as trapped ascospores had low abundance, while pycnidia and conidia were highly abundant. Both P. citricarpa and endophytic Phyllosticta spp. were identified, with P. citricarpa being dominant. In replicated field trials, we determined that infection of Imperial mandarin fruit by P. citricarpa occurred from fruit set until week 20 of fruit development, with the key infection events taking place between weeks 4 and 16 in Queensland subtropical conditions. These results demonstrate that protecting fruit during weeks 4 to 16 significantly reduced P. citricarpa infection. We found no significant correlation between the disease incidence in fruit and P. citricarpa conidial abundance in leaf litter or ascospore abundance measured by volumetric spore trapping. Therefore, it is suggested that inoculum sources in the tree canopy other than those detected by spore trapping and direct leaf litter monitoring may play a major role in the epidemiology of citrus black spot. Improved knowledge regarding epidemiology of P. citricarpa and an understanding of propagules causing infection may aid in development of more effective disease management strategies.

Microsporidia Nosema spp. – obligate bee parasites are transmitted by air

Microsporidia Nosema are transferred among bees via the faecal-oral route. Nosema spp. spores have been detected on flowers and transferred to hives along with the bee pollen. The aim of the present study was to determine whether Nosema microsporidia are transferred by air in an apiary, in a control area (without the presence of bee colonies), and/or in a laboratory during cage experiments with artificially infected bees. The novel way of transmission by air was investigated by the volumetric method using a Hirst-type aerobiological sampler located on the ground in the apiary, in the Botanical Garden and on the laboratory floor. Concurrently, the mean rate of Nosema infections in the foragers in the apiary was estimated with the Bürker haemocytometer method. Spore-trapping tapes were imaged by means of light microscopy, Nomarski interference contrast microscopy and scanning electron microscopy. The highest concentration of Nosema spores per 1m3 of air (4.65) was recorded in August, while the lowest concentration (2.89) was noted in July. This was confirmed by a Real-Time PCR analysis. The presence of N. apis as well as N. ceranae was detected in each of the tested tapes from the apiary. The average copy number of N. apis was estimated at 14.4 × 104 copies per 1 cm2 of the tape; whereas the number of N. ceranae was 2.24 × 104 copies per tape per 1 cm2. The results indicate that Nosema microsporidia were transferred by the wind in the apiary, but not in the Botanical Garden and laboratory by air. This was confirmed by genetic analyses. DNA from immobilised biological material was isolated and subjected to a PCR to detect the Nosema species. A fragment of the 16S rRNA gene, characteristic of Nosema apis and N. ceranae, was detected. Our research adds knowledge about the transfer of Nosema spp. microsporidia in the natural environment and indicates the season associated with the greatest risk of a bee colony infection with Nosema spp.

Identification, Pathogenicity, and Spore Trapping of Colletotrichum karstii Associated with Twig and Shoot Dieback in California

Colletotrichum Corda, 1831 species are well-documented pathogens of citrus that are associated with leaf and fruit anthracnose diseases. However, their role in twig and shoot dieback diseases of citrus has recently become more prominent. Recent surveys of orchards in the Central Valley of California have revealed C. gloeosporioides and a previously undocumented species, C. karstii, to be associated with twig and shoot dieback. Pathogenicity tests using clementine (cv. 4B) indicated that both C. karstii and C. gloeosporioides are capable of producing lesions following inoculation of citrus stems. Pathogenicity tests also revealed C. karstii to be the most aggressive fungal species producing the longest lesions after 15 months. The majority of spores trapped during this study were trapped during or closely following a precipitation event with the majority of spores being trapped from January through May. These findings confirm C. karstii as a new pathogen of citrus in California.

Epidemiology of Exobasidium Leaf and Fruit Spot of Rabbiteye Blueberry: Pathogen Overwintering, Primary Infection, and Disease Progression on Leaves and Fruit

Epidemiological field studies utilizing disease monitoring, spore trapping, and trap plants were conducted on rabbiteye blueberry (Vaccinium virgatum) between 2014 and 2017 to shed light on the epidemiology of Exobasidium leaf and fruit spot, an emerging disease in the southeastern United States caused by the fungus Exobasidium maculosum. Wash plating of field-collected blueberry tissue from the late dormant season through bud expansion showed that the pathogen overwintered epiphytically on blueberry plants in the field, most likely in its yeast-like conidial stage. Agrichemical applications during the dormant season altered epiphytic populations of the pathogen, which correlated directly with leaf spot incidence later in the spring. Disease monitoring of field plants and weekly exposure of potted trap plants revealed that young leaves at the mouse-ear stage were most susceptible to infection, that disease incidence on leaves progressed monocyclically, and that infection periods were associated with rainfall variables such as the number of days per week with ≥1.0 mm of rain or cumulative weekly rainfall. Weekly spore trapping with an Andersen sampler showed that airborne inoculum was detected only after sporulating leaf lesions producing basidiospores were present in the field, suggesting that the primary inoculum is not airborne. The first symptoms on young, green fruit were observed soon after petal fall (requiring removal of the waxy fruit layer to visualize lesions), and visible disease progress on fruit was delayed by 1 to 3 weeks relative to that on leaves. Fruit infection of field plants and trap plants occurred before airborne propagules were detected by spore trapping and before sporulating leaf lesions were present in the field. Hence, this study showed that fruit infections are initiated by the same initial inoculum as leaf infections but it was not possible to conclusively exclude the possibility of a contribution of basidiospore inoculum from leaf lesions to disease progress on later developing fruit. This is one of only a few studies addressing the epidemiology and disease cycle of an Exobasidium sp. in a pathosystem where artificial inoculation has not been possible to date.