The effects of chemical fungicides and salicylic acid on the apple microbiome and fungal disease incidence under changing environmental conditions.

Epiphytic and endophytic micro-organisms associated with plants form complex communities on or in their host plant. These communities influence physiological traits, development, and host susceptibility to abiotic and biotic stresses, and these communities are theorized to have evolved alongside their hosts, forming a unit of selection known as the holobiont. The microbiome is highly variable and can be influenced by abiotic factors, including applied exogenous agents. In this study, we compared the impact of chemical fungicide and salicylic acid treatments on the fungal communities of "Honeycrisp" apples at harvest over two consecutive growing years. We demonstrated variations in fungal community structure and composition by tissue type, growing season, and treatment regimes and that fungicide treatments were associated with reduced network complexity. Finally, we show that the inclusion of salicylic acid with 50% less chemical fungicides in an integrated spray program allowed a reduction in fungicide use while maintaining effective control of disease at harvest and following storage.

Fungal Endophytes: Discovering What Lies within Some of Canada's Oldest and Most Resilient Grapevines.

Plant diseases and pests reduce crop yields, accounting for global crop losses of 30% to 50%. In conventional agricultural production systems, these losses are typically controlled by applying chemical pesticides. However, public pressure is mounting to curtail agrochemical use. In this context, employing beneficial endophytic microorganisms is an increasingly attractive alternative to the use of conventional chemical pesticides in agriculture. A multitude of fungal endophytes are naturally present in plants, producing enzymes, small peptides, and secondary metabolites due to their bioactivity, which can protect hosts from pathogens, pests, and abiotic stresses. The use of beneficial endophytic microorganisms in agriculture is an increasingly attractive alternative to conventional pesticides. The aim of this study was to characterize fungal endophytes isolated from apparently healthy, feral wine grapes in eastern Canada that have grown without agrochemical inputs for decades. Host plants ranged from unknown seedlings to long-lost cultivars not widely propagated since the 1800s. HPLC-MS was used to identify unique endophyte-derived chemical compounds in the host plants, while dual-culture competition assays showed a range in endophytes' ability to suppress the mycelial growth of Botrytis, which is typically controlled in viticulture with pesticides. Twelve of the most promising fungal endophytes isolated were identified using multilocus sequencing and morphology, while DNA barcoding was employed to identify some of their host vines. These fungal endophyte isolates, which consisted of both known and putative novel strains, belonged to seven genera in six families and five orders of Ascomycota. Exploring the fungal endophytes in these specimens may yield clues to the vines' survival and lead to the discovery of novel biocontrol agents.

The apocarotenoid ß-ionone regulates the transcriptome of Arabidopsis thaliana and increases its resistance against Botrytis cinerea.

Carotenoids are isoprenoid pigments indispensable for photosynthesis. Moreover, they are the precursor of apocarotenoids, which include the phytohormones abscisic acid (ABA) and strigolactones (SLs) as well as retrograde signaling molecules and growth regulators, such as ß-cyclocitral and zaxinone. Here, we show that the application of the volatile apocarotenoid ß-ionone (ß-I) to Arabidopsis plants at micromolar concentrations caused a global reprogramming of gene expression, affecting thousands of transcripts involved in stress tolerance, growth, hormone metabolism, pathogen defense, and photosynthesis. This transcriptional reprogramming changes, along with induced changes in the level of the phytohormones ABA, jasmonic acid, and salicylic acid, led to enhanced Arabidopsis resistance to the widespread necrotrophic fungus Botrytis cinerea (B.c.) that causes the gray mold disease in many crop species and spoilage of harvested fruits. Pre-treatment of tobacco and tomato plants with ß-I followed by inoculation with B.c. confirmed the effect of ß-I in increasing the resistance to this pathogen in crop plants. Moreover, we observed reduced susceptibility to B.c. in fruits of transgenic tomato plants overexpressing LYCOPENE ß-CYCLASE, which contains elevated levels of endogenous ß-I, providing a further evidence for its effect on B.c. infestation. Our work unraveled ß-I as a further carotenoid-derived regulatory metabolite and indicates the possibility of establishing this natural volatile as an environmentally friendly bio-fungicide to control B.c.

Why Do We Need Alternative Methods for Fungal Disease Management in Plants?

Fungal pathogens pose a major threat to food production worldwide. Traditionally, chemical fungicides have been the primary means of controlling these pathogens, but many of these fungicides have recently come under increased scrutiny due to their negative effects on the health of humans, animals, and the environment. Furthermore, the use of chemical fungicides can result in the development of resistance in populations of phytopathogenic fungi. Therefore, new environmentally friendly alternatives that provide adequate levels of disease control are needed to replace chemical fungicides-if not completely, then at least partially. A number of alternatives to conventional chemical fungicides have been developed, including plant defence elicitors (PDEs); biological control agents (fungi, bacteria, and mycoviruses), either alone or as consortia; biochemical fungicides; natural products; RNA interference (RNAi) methods; and resistance breeding. This article reviews the conventional and alternative methods available to manage fungal pathogens, discusses their strengths and weaknesses, and identifies potential areas for future research.

First Report of Diplodia intermedia Causing Canker and Dieback Diseases on Apple Trees in Canada.

A dieback of apple trees (Malus domestica (Suckow) Borkh.) associated with cankers was observed in commercial orchards in southwestern Ontario, Canada, in 2019. Fifteen 2 to 10-year-old symptomatic trees were collected from three orchards exhibiting up to 37% disease incidence. Small sections of diseased wood (1 cm long) were surface sterilized with 70% ethanol for 30 sec and 1% NaClO for 20 min, rinsed thrice in sterile water, placed on 2% PDA (Difco) amended with kanamycin (50 mg liter-1), and incubated at 22°C for 5 days in the dark (Ilyukhin et al. 2023). Fungal colonies that were consistently isolated were hyphal-tipped, transferred to individual PDA plates and incubated at 22°C for 7 days in the dark. Purified isolates with same characteristics were classed into morphotypes. One morphotype was initially white and turned dark olivaceous with dense aerial mycelium. Pycnidia were produced on pine needles on PDA (Fig. S2) after incubation at 22°C for 17 days in the dark. Conidia were brown, aseptate, ovoid, and measured 27.9 to 31.3 µm x 12.1 to 14.2 µm (mean ± S.D. of 15 conidia = 29.9 ± 0.9 µm × 13.2 ± 0.6 µm), the typical morphology of a Diplodia sp. (Phillips et al. 2012). Genomic DNA was extracted from a 7-day-old culture of a representative isolate M45-28, using the Plant/Fungi DNA Isolation Kit (Norgen Biotech, Canada). The internal transcribed spacer (ITS), translation elongation factor 1-α (EF1-α) and ß-tubulin gene regions were amplified and sequenced with primers ITS1/ITS4, EF1-728F/EF1-986R and Bt2a/Bt2b and deposited in GenBank with accession numbers MZ970605, MZ995430 and MZ995431, respectively. Based on the sequence, the fungus was identified as Diplodia intermedia A.J.L. Phillips et al. and matched isolates from different hosts and countries (ITS: 100%, MG220378; EF1-α: 100%, MG220385; ß-tubulin: 99.24%, MT592502). The maximum likelihood-based phylogenetic analysis of ITS, EF1-α and ß-tubulin concatenated sequences was performed using IQ-Tree 2.2.2.7 (Minh et al. 2020). M45-28 was clustered with high bootstrap support values with D. intermedia isolates from the Westerdijk Fungal Biodiversity Institute collection, including the ex-holotype (CBS 124462) (Fig. S1). A living culture of M45-28 was deposited in the Canadian Collection of Fungal Cultures (DAOMC 252253). Pathogenicity assay was conducted by inoculating mycelial plugs from a 7-day-old culture of M45-28 into wounds made on the trunk of 5 eight-month-old potted healthy 'Royal Gala' apple seedlings. Five control seedlings were inoculated with sterile plugs. Canker symptoms appeared 15 days after inoculation, spread around, up and down the main stem from the inoculation point, and by 7 weeks the upper portion of the seedling was dead (Fig. S2). Diplodia intermedia was re-isolated from all inoculated seedlings and species identity was confirmed by sequencing as described above, fulfilling Koch's postulates. Control seedlings remained symptomless and the fungus was not isolated from the wood. Diplodia intermedia was reported to cause cankers on apple in Uruguay (Delgado-Cerrone et al. 2016), wild apple (Malus sylvestris) in Portugal (Phillips et al. 2012), grapevines in France (Comont et al. 2016) and forest trees in Iran (Kazemzadeh Chakusary et al. 2019). To the best of our knowledge, this is the first report of D. intermedia causing canker and dieback diseases on apple trees in Canada. Further studies are required to better understand the epidemiology involved in the dynamic spread of the disease in order to recommend an adequate phytosanitary program for its control.

Apple Root Microbiome as Indicator of Plant Adaptation to Apple Replant Diseased Soils.

The tree fruit industry in Nova Scotia, Canada, is dominated by the apple (Malus domestica) sector. However, the sector is faced with numerous challenges, including apple replant disease (ARD), which is a well-known problem in areas with intensive apple cultivation. A study was performed using 16S rRNA/18S rRNA and 16S rRNA/ITS2 amplicon sequencing to assess soil- and root-associated microbiomes, respectively, from mature apple orchards and soil microbiomes alone from uncultivated soil. The results indicated significant (p < 0.05) differences in soil microbial community structure and composition between uncultivated soil and cultivated apple orchard soil. We identified an increase in the number of potential pathogens in the orchard soil compared to uncultivated soil. At the same time, we detected a significant (p < 0.05) increase in relative abundances of several potential plant-growth-promoting or biocontrol microorganisms and non-fungal eukaryotes capable of promoting the proliferation of bacterial biocontrol agents in orchard soils. Additionally, the apple roots accumulated several potential PGP bacteria from Proteobacteria and Actinobacteria phyla, while the relative abundances of fungal taxa with the potential to contribute to ARD, such as Nectriaceae and plant pathogenic Fusarium spp., were decreased in the apple root microbiome compared to the soil microbiome. The results suggest that the health of a mature apple tree can be ascribed to a complex interaction between potential pathogenic and plant growth-promoting microorganisms in the soil and on apple roots.

Performance Optimization of Wearable Printed Human Body Temperature Sensor Based on Silver Interdigitated Electrode and Carbon-Sensing Film.

The human body's temperature is one of the most important vital markers due to its ability to detect various diseases early. Accurate measurement of this parameter has received considerable interest in the healthcare sector. We present a novel study on the optimization of a temperature sensor based on silver interdigitated electrodes (IDEs) and carbon-sensing film. The sensor was developed on a flexible Kapton thin film first by inkjet printing the silver IDEs, followed by screen printing a sensing film made of carbon black. The IDE finger spacing and width of the carbon film were both optimized, which considerably improved the sensor's sensitivity throughout a wide temperature range that fully covers the temperature of human skin. The optimized sensor demonstrated an acceptable temperature coefficient of resistance (TCR) of 3.93 × 10-3 °C-1 for temperature sensing between 25 °C and 50 °C. The proposed sensor was tested on the human body to measure the temperature of various body parts, such as the forehead, neck, and palm. The sensor showed a consistent and reproducible temperature reading with a quick response and recovery time, exhibiting adequate capability to sense skin temperatures. This wearable sensor has the potential to be employed in a variety of applications, such as soft robotics, epidermal electronics, and soft human-machine interfaces.

Impacts of abiotic factors on the fungal communities of 'Honeycrisp' apples in Canada.

The maintenance of the beneficial plant microbiome to control plant pathogens is an emerging concept of disease management, and necessitates a clear understanding of these microbial communities and the environmental factors that affect their diversity and compositional structure. As such, studies investigating the microbiome of economically significant cultivars within each growing region are necessary to develop adequate disease management strategies. Here, we assessed the relative impacts of growing season, management strategy, and geographical location on the fungal microbiome of 'Honeycrisp' apples from seven different orchard locations in the Atlantic Maritime Ecozone for two consecutive growing years. Though apple fruit tissue was dominated by relatively few fungal genera, significant changes in their fungal communities were observed as a result of environmental factors, including shifts in genera with plant-associated lifestyles (symbionts and pathogens), such as Aureobasidium, Alternaria, Penicillium, Diplodia, and Mycosphaerella. Variation in fungal composition between different tissues of fruit was also observed. We demonstrate that growing season is the most significant factor affecting fungal community structure and diversity of apple fruit, suggesting that future microbiome studies should take place for multiple growing seasons to better represent the host-microbiome of perennial crops under different environmental conditions.

The Arabidopsis D27-LIKE1 is a cis/cis/trans-ß-carotene isomerase that contributes to Strigolactone biosynthesis and negatively impacts ABA level.

The enzyme DWARF27 (D27) catalyzes the reversible isomerization of all-trans- into 9-cis-ß-carotene, initiating strigolactone (SL) biosynthesis. Genomes of higher plants encode two D27-homologs, D27-like1 and -like2, with unknown functions. Here, we investigated the enzymatic activity and biological function of the Arabidopsis D27-like1. In vitro enzymatic assays and expression in Synechocystis sp. PCC6803 revealed an unreported 13-cis/15-cis/9-cis- and a 9-cis/all-trans-ß-carotene isomerization. Although disruption of AtD27-like1 did not cause SL deficiency phenotypes, overexpression of AtD27-like1 in the d27 mutant restored the more-branching phenotype, indicating a contribution of AtD27-like1 to SL biosynthesis. Accordingly, generated d27 d27like1 double mutants showed a more pronounced branching phenotype compared to d27. The contribution of AtD27-like1 to SL biosynthesis is likely a result of its formation of 9-cis-ß-carotene that was present at higher levels in AtD27-like1 overexpressing lines. By contrast, AtD27-like1 expression correlated negatively with the content of 9-cis-violaxanthin, a precursor of ABA, in shoots. Consistently, ABA levels were higher in shoots and also in dry seeds of the d27like1 and d27 d27like1 mutants. Transgenic lines expressing GUS driven by the AtD27LIKE1 promoter and transcript analysis of hormone-treated Arabidopsis seedlings revealed that AtD27LIKE1 is expressed in different tissues and affects ABA and auxin. Taken together, our work reports a cis/cis-ß-carotene isomerase that affects the content of both cis-carotenoid-derived plant hormones, ABA and SLs.

Effects of Temperature and Wetness on Components of the Infection Cycle of Valdensia heterodoxa in Lowbush Blueberry.

Valdensia leaf spot, caused by Valdensia heterodoxa, is a serious disease of lowbush blueberry. The disease may develop rapidly, resulting in extensive defoliation of fields. The purpose of this study was to examine the effects of temperature and wetness duration on various components of the infection cycle to gain a better understanding of epidemic development that might lead to improved management practices. Lesions on leaves appeared 6 h after inoculation at 20°C and were larger on young 3-week-old leaves compared with 8-week-old leaves. Incidence of infection on 3-week-old leaves was lowest at 5°C, highest at 15 and 20°C, and failed to occur at 30°C. Defoliation began 48 h after inoculation at 20 and 25°C but was slower at higher and lower temperatures. Conidia production and release from colonized leaves began 48 h after inoculation at 15 and 19°C. Total conidia production was lowest at 7°C, highest at 15°C, and progressively declined at 19 and 23°C. Production of conidia lasted 2 to 3 days. Sclerotia formed mainly along the midveins and were similar in size at 5 to 15°C, largest at 20°C, and smallest at 25°C. Conidia formed directly on sclerotia that were overwintered outdoors and then incubated on moist filter paper. Conidia production began after 48 h at 10, 15, and 20°C. Total production was lowest at 5°C, highest at 20°C, failed to occur at 25°C, and ceased after 10 days at all temperatures. These data show that at optimal temperatures, relatively short wet periods are required for conidia production on overwintered sclerotia, infection of leaves, and subsequent conidia production on diseased leaves that may account for the sudden and rapid spread of disease in fields. The data will be useful for helping growers identify weather conditions favorable for disease development.

Canonical strigolactones are not the major determinant of tillering but important rhizospheric signals in rice.

Strigolactones (SLs) are a plant hormone inhibiting shoot branching/tillering and a rhizospheric, chemical signal that triggers seed germination of the noxious root parasitic plant Striga and mediates symbiosis with beneficial arbuscular mycorrhizal fungi. Identifying specific roles of canonical and noncanonical SLs, the two SL subfamilies, is important for developing Striga-resistant cereals and for engineering plant architecture. Here, we report that rice mutants lacking canonical SLs do not show the shoot phenotypes known for SL-deficient plants, exhibiting only a delay in establishing arbuscular mycorrhizal symbiosis, but release exudates with a significantly decreased Striga seed-germinating activity. Blocking the biosynthesis of canonical SLs by TIS108, a specific enzyme inhibitor, significantly lowered Striga infestation without affecting rice growth. These results indicate that canonical SLs are not the determinant of shoot architecture and pave the way for increasing crop resistance by gene editing or chemical treatment.

Fungal Endophytes and Their Role in Agricultural Plant Protection against Pests and Pathogens.

Virtually all examined plant species harbour fungal endophytes which asymptomatically infect or colonize living plant tissues, including leaves, branches, stems and roots. Endophyte-host interactions are complex and span the mutualist-pathogen continuum. Notably, mutualist endophytes can confer increased fitness to their host plants compared with uncolonized plants, which has attracted interest in their potential application in integrated plant health management strategies. In this review, we report on the many benefits that fungal endophytes provide to agricultural plants against common non-insect pests such as fungi, bacteria, nematodes, viruses, and mites. We report endophytic modes of action against the aforementioned pests and describe why this broad group of fungi is vitally important to current and future agricultural practices. We also list an extensive number of plant-friendly endophytes and detail where they are most commonly found or applied in different studies. This review acts as a general resource for understanding endophytes as they relate to potential large-scale agricultural applications.

Effect of RH, Temperature, Light, and Plant Age on Infection of Lowbush Blueberry by Sphaerulina vaccinii.

Leaf spot and stem canker caused by Sphaerulina vaccinii is associated with premature defoliation in lowbush blueberry resulting in reduced yields. In this study, we investigated the impact of free water, RH, temperature, light, and plant age on leaf infection under controlled conditions. On potato dextrose agar, germination of conidia was usually polar. Growth was minimal at 5 and 10°C, increased at 15 and 20°C, was maximal at 25°C and decreased at 30°C. Percentage of germinated conidia on inoculated blueberry leaves incubated in dark controlled-humidity chambers for 3 days (25°C) was 86.0, 90.5, 81.3, and 28.3 in free water, 100, 97.5 or 95% RH, respectively. Germination did not occur at 90 or 85% RH. Infection of inoculated plants, however, was not favored by free water, but rather by high RH (>95%) and a 14-h photoperiod (180 µmol/m2 per second). Infection failed in continuous darkness, continuous light, or continuous darkness followed by 4, 8, or 12 h of light. Light and scanning electron microscopy showed that hyphal penetration into stomata on abaxial leaf surfaces was strongly tropic. When germ tubes grew in close proximity to a stomate, a penetration hypha formed at ∼90° angles to the germ tube and took the closest path to the stomate. Stomatal penetration was usually direct, but occasionally appressorium-like hyphal swellings formed over stomatal openings. When inoculated plants were exposed to high RH (>95%) at various temperatures, infection occurred after 4 days at 10°C, after 3 days at 15°C and after 1 day at 20 and 25°C. Infection failed to occur at 30°C. Disease severity also increased with duration of the humid period. When leaves were examined microscopically, those that had been incubated for 6 days showed a substantially greater network of epiphytic growth with more stomatal penetrations compared with those incubated for 3 days. Infection was substantially reduced when the humid period was interrupted by alternating days of low RH (60%). Two-week-old leaves were 2.7 times more susceptible than 8-week-old leaves.

An alternative, zeaxanthin epoxidase-independent abscisic acid biosynthetic pathway in plants.

Abscisic acid (ABA) is an important carotenoid-derived phytohormone that plays essential roles in plant response to biotic and abiotic stresses as well as in various physiological and developmental processes. In Arabidopsis, ABA biosynthesis starts with the epoxidation of zeaxanthin by the ABA DEFICIENT 1 (ABA1) enzyme, leading to epoxycarotenoids; e.g., violaxanthin. The oxidative cleavage of 9-cis-epoxycarotenoids, a key regulatory step catalyzed by 9-CIS-EPOXYCAROTENOID DIOXYGENASE, forms xanthoxin, which is converted in further reactions mediated by ABA DEFICIENT 2 (ABA2), ABA DEFICIENT 3 (ABA3), and ABSCISIC ALDEHYDE OXIDASE 3 (AAO3) into ABA. By combining genetic and biochemical approaches, we unravel here an ABA1-independent ABA biosynthetic pathway starting upstream of zeaxanthin. We identified the carotenoid cleavage products (i.e., apocarotenoids, ß-apo-11-carotenal, 9-cis-ß-apo-11-carotenal, 3-OH-ß-apo-11-carotenal, and 9-cis-3-OH-ß-apo-11-carotenal) as intermediates of this ABA1-independent ABA biosynthetic pathway. Using labeled compounds, we showed that ß-apo-11-carotenal, 9-cis-ß-apo-11-carotenal, and 3-OH-ß-apo-11-carotenal are successively converted into 9-cis-3-OH-ß-apo-11-carotenal, xanthoxin, and finally into ABA in both Arabidopsis and rice. When applied to Arabidopsis, these ß-apo-11-carotenoids exert ABA biological functions, such as maintaining seed dormancy and inducing the expression of ABA-responsive genes. Moreover, the transcriptomic analysis revealed a high overlap of differentially expressed genes regulated by ß-apo-11-carotenoids and ABA, suggesting that ß-apo-11-carotenoids exert ABA-independent regulatory activities. Taken together, our study identifies a biological function for the common plant metabolites, ß-apo-11-carotenoids, extends our knowledge about ABA biosynthesis, and provides new insights into plant apocarotenoid metabolic networks.

Iso-anchorene is an endogenous metabolite that inhibits primary root growth in Arabidopsis.

Carotenoid-derived regulatory metabolites and hormones are generally known to arise through the oxidative cleavage of a single double bond in the carotenoid backbone, which yields mono-carbonyl products called apocarotenoids. However, the extended conjugated double bond system of these pigments predestines them also to repeated cleavage forming dialdehyde products, diapocarotenoids, which have been less investigated due to their instability and low abundance. Recently, we reported on the short diapocarotenoid anchorene as an endogenous Arabidopsis metabolite and specific signaling molecule that promotes anchor root formation. In this work, we investigated the biological activity of a synthetic isomer of anchorene, iso-anchorene, which can be derived from repeated carotenoid cleavage. We show that iso-anchorene is a growth inhibitor that specifically inhibits primary root growth by reducing cell division rates in the root apical meristem. Using auxin efflux transporter marker lines, we also show that the effect of iso-anchorene on primary root growth involves the modulation of auxin homeostasis. Moreover, by using liquid chromatography-mass spectrometry analysis, we demonstrate that iso-anchorene is a natural Arabidopsis metabolite. Chemical inhibition of carotenoid biosynthesis led to a significant decrease in the iso-anchorene level, indicating that it originates from this metabolic pathway. Taken together, our results reveal a novel carotenoid-derived regulatory metabolite with a specific biological function that affects root growth, manifesting the biological importance of diapocarotenoids.

Global analysis of the apple fruit microbiome: are all apples the same?

We present the first worldwide study on the apple (Malus × domestica) fruit microbiome that examines questions regarding the composition and the assembly of microbial communities on and in apple fruit. Results revealed that the composition and structure of the fungal and bacterial communities associated with apple fruit vary and are highly dependent on geographical location. The study also confirmed that the spatial variation in the fungal and bacterial composition of different fruit tissues exists at a global level. Fungal diversity varied significantly in fruit harvested in different geographical locations and suggests a potential link between location and the type and rate of postharvest diseases that develop in each country. The global core microbiome of apple fruit was represented by several beneficial microbial taxa and accounted for a large fraction of the fruit microbial community. The study provides foundational information about the apple fruit microbiome that can be utilized for the development of novel approaches for the management of fruit quality and safety, as well as for reducing losses due to the establishment and proliferation of postharvest pathogens. It also lays the groundwork for studying the complex microbial interactions that occur on apple fruit surfaces.

First Report of Moldy Core of Sweet Tango Apples from New Zealand Caused by Alternaria arborescens.

Moldy core is a fungal disease of apple fruits that is characterized by mycelial growth in the seed locules and is sometimes accompanied by penetration of the immediate surrounding flesh. The disease can go undetected until the fruit is cut open, as no external symptoms appear on the fruit. Alternaria, Aspergillus, Cladosporium, Coniothyrium, Epicoccum, Phoma and Stemphylium are some of the common pathogens associated with moldy core (Serdani et al. 2002; Gao et al. 2013; McLeod 2014). The disease is more common in apple cultivars with an open calyx, where spores may initiate infections during the growing season or at the post-harvest storage stage (Spotts et al. 1988). In 2018, a shipment of 'Sweet Tango' apples from New Zealand to Scotian Gold Co-operative Ltd., Nova Scotia, Canada, was found to be affected by moldy core. Moderate to severe moldy core symptoms were observed when 10 apples were cut open (Figure S1). In comparison, 'Sweet Tango' apples grown in Nova Scotia showed no moldy core symptoms when 10 random fruits were cut open. Small pieces of the diseased fruit tissue from the core region were surface-disinfected for 1 min in 1% NaOCl, rinsed three times with sterilized water and placed onto potato dextrose agar (PDA) dishes. The PDA dishes were incubated in dark at 22 oC and single spore isolation was carried out to fresh PDA dishes. These isolate produced colonies of regular shape, tan black with prominent white gray margin and gray colour conidia (Figure S2 AB). The colonies turn dark black after 3 weeks of growth on PDA. Mycelia were septate and conidia were oval or obclavate or club-shaped with a tapering end with 4-6 longitudinal and transverse septa (Figure S2 C-D). The size of conidia ranges from 12.5-20 x 8.7-12.5 µM on 20 days old PDA dishes. Based on the size and shape of conidia and other morphological characteristics the isolated fungi were identical to Alternaria spp. (Simmons 2007). To assess the identity of the isolated pathogen species by multi-locus sequence analysis, genomic DNA was extracted from the pure cultures of two isolates (5.8 and 8) using the E.Z.N.A. SP Fungal DNA Kit (Omega Bio-Tek). The glyceraldehyde-3-phosphate dehydrogenase (GAPDH), major allergen (Alt a 1), OPA10-2, the internal transcribed spacer (ITS) region of ribosomal DNA and the translation elongation factor 1-α (TEF1-α) region from two Alternaria spp. isolates (5.8 and 8) were amplified and sequenced using primers gpd1/2 (Berbee et al. 1999), A21F/A21R (Gabriel 2015), OPA10-2/ OPA10-2L (Andrew et al. 2009), ITS1/ITS4 (White et al. 1990) and EF1-up /EF1-low (O'Donnell et al. 1998) respectively. The resulting sequences of both isolates were deposited in the NCBI GenBank (GAPDH; MW411052, MW411053, Alt a 1; MW411050, MW411051, OPA10-2; MW415762, MW415763, ITS; MK140445, MT225559, TEF1-α; MT305773 and MT305774 ). Sequences of GAPDH, Alt a 1, OPA-10-2, ITS and TEF1-α genes of both isolates were identical to each other and showed 100 %, 100 %, 99.21 %, 100% and 100% identity to A. arborescens S. (AY278810.1, AY563303.1, KP124712.1, KY965831.1, KY965831.1) respectively. Identity with reference strain CBS 102605 confirms that both of the isolated strains 5.8 and 8 are A. arborescens. The pathogenicity of the two A. arborescens isolates were confirmed by artificially inoculating healthy 'Sweet Tango' fruit by dispensing the conidial suspension directly on the seed locule. Briefly, surface-disinfected fruits were air-dried for 5 min and then peeled using a sterilized knife and cut transversally. Each half of the fruit was inoculated with 100 µl of conidial suspensions (∼1 × 104 conidia/ml) in potato dextrose broth (PDB) and incubated at 22 °C in a humid chamber for 7-10 days, or until symptoms with visible mycelial growth were observed. The control fruits were treated with 100 µl of sterilized PDB. Both A. arborescens isolates produced visible moldy core symptoms on the inoculated 'Sweet Tango' fruits, whereas no symptoms were observed on the control fruits (Figure S1). The experiment was repeated three times with at least three replicates with similar results. A. arborescens was successfully re-isolated from the artificially-inoculated fruits to complete Koch's postulates. To our knowledge, this is the first report of Alternaria arborescens causing moldy core disease in 'Sweet Tango' apples from New Zealand. Acknowledgments We thank Eric Bevis for his help in sample preparation for DNA sequencing, Willy Renderos for pathogenicity assay. We also thank Joan Hebb (Scotian Gold Cooperative Ltd.,) for providing the apple sample for this study. This research was made possible through financial support from Agriculture and Agri-Food Canada. The authors(s) declare no conflict of interest. Literature Cited Andrew M., Peever T.L., Pryor B.M. An expanded multilocus phylogeny does not resolve species among the small-spored Alternaria species complex. 2009. Mycologia. 101:95-109. Berbee, M. L. et al. 1999. Cochliobolus phylogenetics and the origin of known, highly virulent pathogens, inferred from ITS and glyceraldehyde-3-phosphate dehydrogenase gene sequences Mycologia. 91:964. Gabriel, M.F. I. Postigo, A. Gutiérrez-Rodríguez, E. Suñén, C.T. Tomaz, J. Martínez 2015. Development of a PCR-based tool for detecting immunologically relevant Alt a 1 and Alt a 1 homologue coding sequences. Medical Mycology. 53 (6):636-642. Gao, L. L., Zhang, Q., Sun, X. Y., Jiang, L., Zhang, R., Sun, G. Y., Zha, Y. L., and Biggs, A. R. 2013. Etiology of moldy core, core browning, and core rot of Fuji apple in China. Plant Dis. 97:510-516. Kerry, O'Donnell, H.C. Kistler, E. Cigelnik, R.C. Ploetz. 1998. Multiple evolutionary origins of the fungus causing Panama disease of banana: concordant evidence from nuclear and mitochondrial gene genealogies. PNAS. 95: 2044-2049. McLeod, A. 2014. Moldy core and core rots. Pages 40-41 in: Compendium of Apple and Pear Diseases and Pests, 2nd ed. T. B. Sutton, H. S. Aldwinckle, A. M. Agnello, and J. F. Walgenbach, eds. American Phytopathological Society, St Paul, MN. Serdani, M., Kang, J. C., Peever, T. L., Andersen, B., and Crous, P. W. 2002. Characterization of Alternaria species groups associated with core rot of apples in South Africa. Mycol. Res. 106:561-569. Simmons, E. G. 2007. Alternaria: an identification manual. CBS Biodiversity Series. 6:780 pp. Spotts, R. A., Holmes, R. J., and Washington, W. S. 1988. Factors affecting wet core rot of apples. Australas. Plant Pathol. 17:53-57. White, T. J., 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. M. A. Innis, D. H. Gelfand, J. J. Sninsky, and T. J. White, eds. San Diego, CA: Academic Press. Woudenberg, J. H. C., et al. 2015. Alternaria section Alternaria: Species, formae speciales or pathotypes. Stud. Mycol. 82:1-21.

Identification and Characterization of Sphaerulina vaccinii sp. nov. as the Cause of Leaf Spot and Stem Canker in Lowbush Blueberry and Its Epidemiology.

Septoria leaf spot and stem canker is an important disease of lowbush blueberry, but the causal pathogen has not been accurately identified. Based on sequence analysis of the internal transcribed spacer, translation elongation factor 1 alpha, RNA polymerase II second largest subunit, 28S nuclear ribosomal DNA gene, and ß-tubulin genes, the pathogen aligns closely with the genus Sphaerulina. The phylogenetic analyses based on these loci demonstrate that while the pathogen is closely related to the species Sphaerulina amelanchier, it is sufficiently distinct to warrant a new species designation. No ascomata of the teleomorph were found; however, ascospores recovered from leaves fit, morphologically, with the genus Mycosphaerella. The morphological data also support a new species designation. Based on the host that this pathogen infects, we propose the name as Sphaerulina vaccinii and the disease as Sphaerulina leaf spot and stem canker. Under field conditions, it appears that initial inoculum originates from pycnidia on overwintered leaves and stem lesions (cankers) on fruiting stems. More than 90% of the initial inoculum was released during the flowering period from late May through June. Leaf spots began to appear in early June and disease severity increased in a linear manner over time. Secondary inoculum production from diseased foliage was minimal and not considered important epidemiologically. Defoliation resulting from disease began in early July and increased in a nonlinear manner thereafter. Manual defoliation of blueberry stems at various times prior to harvest showed the substantial extent to which premature defoliation by this disease can affect yield. Stem lesions were also shown to have an impact on yield, even though stems were not killed.

UhAVR1, an HR-Triggering Avirulence Effector of Ustilago hordei, Is Secreted via the ER-Golgi Pathway, Localizes to the Cytosol of Barley Cells during in Planta-Expression, and Contributes to Virulence Early in Infection.

The basidiomycete Ustilago hordei causes covered smut disease of barley and oats. Virulence effectors promoting infection and supporting pathogen lifestyle have been described for this fungus. Genetically, six avirulence genes are known and one codes for UhAVR1, the only proven avirulence effector identified in smuts to date that triggers complete immunity in barley cultivars carrying resistance gene Ruh1. A prerequisite for resistance breeding is understanding the host targets and molecular function of UhAVR1. Analysis of this effector upon natural infection of barley coleoptiles using teliospores showed that UhAVR1 is expressed during the early stages of fungal infection where it leads to HR triggering in resistant cultivars or performs its virulence function in susceptible cultivars. Fungal secretion of UhAVR1 is directed by its signal peptide and occurs via the BrefeldinA-sensitive ER-Golgi pathway in cell culture away from its host. Transient in planta expression of UhAVR1 in barley and a nonhost, Nicotiana benthamiana, supports a cytosolic localization. Delivery of UhAVR1 via foxtail mosaic virus or Pseudomonas species in both barley and N. benthamiana reveals a role in suppressing components common to both plant systems of Effector- and Pattern-Triggered Immunity, including necrosis triggered by Agrobacterium-delivered cell death inducers.

Comparative genomic analysis of Erwinia amylovora reveals novel insights in phylogenetic arrangement, plasmid diversity, and streptomycin resistance.

Erwinia amylovora is a destructive pathogen of Rosaceous plants and an economic concern worldwide. Herein, we report 93 new E. amylovora genomes from North America, Europe, the Mediterranean, and New Zealand. This new genomic information demonstrates the existence of three primary clades of Amygdaloideae (apple and pear) infecting E. amylovora and suggests all three independently originate from North America. The comprehensive sequencing also identified and confirmed the presence of 7 novel plasmids ranging in size from 2.9 to 34.7 kbp. While the function of the novel plasmids is unknown, the plasmids pEAR27, pEAR28, and pEAR35 encoded for type IV secretion systems. The strA-strB gene pair and the K43R point mutation at codon 43 of the rpsL gene have been previously documented to confer streptomycin resistance. Of the sequenced isolates, rpsL-based streptomycin resistance was more common and was found with the highest frequency in the Western North American clade.