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.

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.

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.

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.

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.

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.

Occurrence of copper-resistant strains and a shift in Xanthomonas spp. causing tomato bacterial spot in Ontario.

Field strains of tomato bacterial spot pathogen (Xanthomonas euvesicatoria, X. vesicatoria, X. perforans, and X. gardneri) were characterized for sensitivity to copper and species composition. A total of 98 strains were isolated from symptomatic leaf and fruit samples collected from 18 tomato fields in Ontario. In greenhouse pathogenicity tests, most of the field strains caused severe (37 strains) to highly severe (23 strains) symptoms on 'Bonny Best' tomato plants, whereas 38 strains caused moderate symptoms. In MGY agar plates amended with various concentrations of copper sulfate, 11 strains were completely sensitive (no growth) and 87 strains were resistant (grew on 1.0 mmol/L or higher copper concentration). PCR analysis of the hrp gene cluster followed by restriction digestion with HaeIII and sequencing identified X. gardneri (35 strains) and X. perforans (26 strains) as predominant species and X. euvesicatoria and X. vesicatoria as less common species in Ontario tomato fields. Separation of field strains into various species was also confirmed with starch hydrolysis activity on agar medium. Moreover, 72 field strains produced shiny greenish-yellow colonies surrounded by a milky zone on xanthomonad differential (Xan-D) medium, and the colonies of 26 strains did not produce a milky zone. Thirty-four strains could not be clustered into any species and 25 of those strains were negative for the hrp gene PCR and also did not produce a milky zone around colonies on Xan-D medium. Our results suggest a widespread existence of copper-resistant strains and an increase in X. perforans strains of bacterial spot pathogen in Ontario. This information on copper resistance and species composition within bacterial spot pathogens in Ontario will be helpful for developing effective disease management strategies, making cultivar selection, and breeding new tomato cultivars.

Isolation, characterization, and formulation of antagonistic bacteria for the management of seedlings damping-off and root rot disease of cucumber.

Antagonistic bacteria are common soil inhabitants with potential to be developed into biofungicides for the management of seedling damping-off, root rot, and other soil-borne diseases of various crops. In this study, antagonistic bacteria were isolated from a commercial potato field and screened for their growth inhibition of fungal and oomycete pathogens in laboratory tests. The biocontrol potential of the 3 most effective antagonistic bacteria from the in vitro tests was evaluated against seedling damping-off and root rot of cucumber caused by Pythium ultimum. Based on phenotypic characteristics, biochemical tests, and sequence analysis of 16S-23S rDNA gene, the 3 antagonistic bacteria were identified as Pseudomonas fluorescens (isolate 9A-14), Pseudomonas sp. (isolate 8D-45), and Bacillus subtilis (isolate 8B-1). All 3 bacteria promoted plant growth and suppressed Pythium damping-off and root rot of cucumber seedlings in growth-room assays. Both pre- and post-planting application of these bacteria to an infested peat mix significantly increased plant fresh masses by 113%-184% and percentage of healthy seedlings by 100%-290%, and decreased damping-off and root rot severity by 27%-50%. The peat and talc formulations of these antagonistic bacteria applied as seed or amendment treatments to the infested peat mix effectively controlled Pythium damping-off and root rot of cucumber seedlings and enhanced plant growth. The survival of all 3 antagonistic bacteria in peat and talc formulations decreased over time at room temperature, but the populations remained above 10(8) CFU/g during the 180-day storage period. The peat formulation of a mixture of 3 bacteria was the best seed treatment, significantly increasing the plant fresh masses by 245% as compared with the Pythium control, and by 61.4% as compared with the noninfested control. This study suggests that the indigenous bacteria from agricultural soils can be developed and formulated as biofungicides for minimizing the early crop losses caused by seedling damping-off and root rot diseases.

Characterization of Fusarium isolates from asparagus fields in southwestern Ontario and influence of soil organic amendments on Fusarium crown and root rot.

Fusarium crown and root rot (FCRR) of asparagus has a complex etiology with several soilborne Fusarium spp. as causal agents. Ninety-three Fusarium isolates, obtained from plant and soil samples collected from commercial asparagus fields in southwestern Ontario with a history of FCRR, were identified as Fusarium oxysporum (65.5%), F. proliferatum (18.3%), F. solani (6.4%), F. acuminatum (6.4%), and F. redolens (3.2%) based on morphological or cultural characteristics and polymerase chain reaction (PCR) analysis with species-specific primers. The intersimple-sequence repeat PCR analysis of the field isolates revealed considerable variability among the isolates belonging to different Fusarium spp. In the in vitro pathogenicity screening tests, 50% of the field isolates were pathogenic to asparagus, and 22% of the isolates caused the most severe symptoms on asparagus. The management of FCRR with soil organic amendments of pelleted poultry manure (PPM), olive residue compost, and fish emulsion was evaluated in a greenhouse using three asparagus cultivars of different susceptibility in soils infested with two of the pathogenic isolates (F. oxysporum Fo-1.5 and F. solani Fs-1.12). Lower FCRR symptom severity and higher plant weights were observed for most treatments on 'Jersey Giant' and 'Grande' but not on 'Mary Washington'. On all three cultivars, 1% PPM consistently reduced FCRR severity by 42 to 96% and increased plant weights by 77 to 152% compared with the Fusarium control treatment. Populations of Fusarium and total bacteria were enumerated after 1, 3, 7, and 14 days of soil amendment. In amended soils, the population of Fusarium spp. gradually decreased while the population of total culturable bacteria increased. These results indicate that soil organic amendments, especially PPM, can decrease disease severity and promote plant growth, possibly by decreasing pathogen population and enhancing bacterial activity in the soil.

Detection of high concentrations of organic acids in fish emulsion and their role in pathogen or disease suppression.

Fish emulsion (FE) added to a sandy-loam soil at 1 and 2% rates reduced the viability of Verticillium dahliae microsclerotia by 39 and 74% in 1 day, 87 and 98% in 3 days, and 95 and 99% in 6 days, respectively. The immediate kill of microsclerotia indicated that FE contains toxic substances. We found in FE high concentrations (400 mmol/liter) of organic acids, including some known toxicants. Glycolic, acetic, formic, n-butyric, and propionic acids were the major organic acids detected in FE at the proportions of 52.5, 26.9, 7.9, 7.2, and 4.7%, respectively. In solution assays, the viability of V. dahliae microsclerotia treated for 24 h in 1, 2, 5, and 10% FE (pH 3.6 to 3.0) or a mixture of organic acids (pH 4.1 to 3.9) equivalent to the proportions in FE was reduced by 74, 94, 97, and 99% or 81, 91, 98, and 99%, respectively. The viability of microsclerotia was increased when the treatment solutions were buffered to pH 6.0. The organic acids mixtures and formic (0.025%) and acetic (0.1%) acids were toxic to Pythium ultimum. A mixture of organic acids (1, 2, and 4%) provided immediate protection of cucumber seedlings from damping-off in P. ultimum-infested muck and sandy-loam soils but not in peat-based mix. FE (1 and 2%) provided immediate protection of cucumber seedlings from damping-off in an infested muck soil, and disease protection was consistent when planting was delayed for 7, 14, and 28 days after adding FE. FE (1, 2, and 4%) did not provide immediate protection of cucumber seedlings from damping-off in a P. ultimum-infested peat-based mix; however, disease suppression was evident when planting was delayed for 7, 14, and 21 days after adding FE. Real-time polymerase chain reaction analyses of the peat-based mix indicated that the P. ultimum populations in the FE-amended mix declined over time. This study suggests that these organic acids in FE played a major role in pathogen or disease suppression, depending on the soil and substrate.

Suppression of Bacterial Spot of Tomato with Foliar Sprays of Compost Extracts Under Greenhouse and Field Conditions.

The efficacy of foliar sprays with compost water extracts (compost extracts) in reducing the severity of bacterial spot of tomato caused by Xanthomonas vesicatoria was investigated. Extracts prepared from composted cow manure, composted pine bark, an organic farm compost, or composted yard waste, applied as foliar sprays on tomato transplants, resulted in a moderate but statistically significant reduction in the severity of bacterial spot. The population of X. vesicatoria in infected leaves was reduced significantly by extracts prepared from composted cow manure. Efficacy of the water extracts was not affected by oxygen concentrations in the suspension during extraction, compost maturity, or sterilization by filtration or autoclaving. The degree of control provided by foliar sprays with the most effective compost extracts did not differ from that obtained with the plant activator acibenzolar-S-methyl. In the field in two growing seasons, foliar sprays with compost water extracts did not reduce the severity of foliar diseases, including bacterial spot. During the 1997 season, when the severity of bacterial spot in the field was high, foliar sprays with compost water extracts significantly reduced the incidence of bacterial spot on tomato fruit. Amending plot soil with several rates of composted yard waste did not lead to additional control of fruit disease over those only sprayed with extracts. Foliar sprays with a mixture of chlorothalonil and copper hydroxide or with acibenzolar-S-methyl reduced the severity of bacterial spot as well as incidence of spot on fruit.

Reduction of Bacterial Spot Disease Severity on Tomato and Pepper Plants with Foliar Applications of Ammonium Lignosulfonate and Potassium Phosphate.

Bacterial spot is a serious and persistent disease problem of tomato and bell pepper in both the United States and Canada. Current disease management practices, based primarily on fixed copper bactericides, do not give consistent, effective protection. Foliar applications of ammonium lignosulfonate (ALS), derived from the wood pulping process, and the fertilizer potassium phosphate (KP) were tested for their ability to control this disease under both greenhouse and field conditions. Acibenzolar-S-methyl was included as a control. Greenhouse-grown tomato transplants treated with acibenzolar-S-methyl, 2 or 4% (vol/vol) ALS, 25 mM KP, or 2% ALS plus 10 mM KP and then inoculated with Xanthomonas campestris pv. vesicatoria had significantly less disease than the unprotected controls. Weekly foliar applications of acibenzolar-S-methyl, ALS, or KP significantly reduced disease severity on the foliage of inoculated field-grown tomato and pepper plants; although less disease appeared on the fruit of these plants, the effect was not always statistically significant except for the acibenzolar-S-methyl treatment. Acibenzolar-S-methyl increased the yield of marketable tomato fruit in 2 of 3 years of the study and that of pepper fruit in 1 of 2 years. There was a marked increase in the yield of marketable fruit on all ALS-treated pepper plants in 2001. None of the treatments significantly increased total tomato or pepper yield. ALS and KP had no observable phytotoxic effect on tomato or pepper foliage. Our results indicate that future integrated disease management programs for bacterial spot may be enhanced by including foliar sprays of these two products.