Evaluation of the Characteristics and Infectivity of the Secondary Inoculum Produced by Plasmopara viticola on Grapevine Leaves by Means of Flow Cytometry and Fluorescence-Activated Cell Sorting.

Plasmopara viticola, the oomycete causing grapevine downy mildew, is one of the most important pathogens in viticulture. P. viticola is a polycyclic pathogen, able to carry out numerous secondary cycles of infection during a single vegetative grapevine season, by producing asexual spores (zoospores) within sporangia. The extent of these infections is strongly influenced by both the quantity (density) and quality (infectivity) of the inoculum produced by the pathogen. To date, the protocols for evaluating all these characteristics are quite limited and time-consuming and do not allow all the information to be obtained in a single run. In this study, a protocol combining flow cytometry (FCM) and fluorescence-activated cell sorting (FACS) was developed to investigate the composition, the infection efficiency and the dynamics of the inoculum produced by P. viticola for secondary infection cycles. In our analyses, we identified different structures within the inoculum, including degenerated and intact sporangia. The latter have been sorted, and single sporangia were directly inoculated on grapevine leaf discs, thus allowing a thorough investigation of the infection dynamics and efficiency. In detail, we determined that, in our conditions, 8% of sporangia were able to infect the leaves and that on a susceptible variety, the time required by the pathogen to reach 50% of total infection is about 10 days. The analytical approach developed in this study could open a new perspective to shed light on the biology and epidemiology of this important pathogen. IMPORTANCE P. viticola secondary infections contribute significantly to the epidemiology of this important plant pathogen. However, the infection dynamics of asexual spores produced by this organism are still poorly investigated. The main challenges in dissecting the grapevine-P. viticola interaction in vitro are attributable to the biotrophic adaptation of the pathogen. This work provides new insights into the infection efficiency and dynamics imputable to P. viticola sporangia, contributing useful information on grapevine downy mildew epidemiology. Moreover, future applications of the sorting protocol developed in this work could yield a significant and positive impact in the study of P. viticola, providing unmatched resolution, precision, and accuracy compared with the traditional techniques.

Characterization of Italian Plasmopara viticola populations for resistance to oxathiapiprolin.

BACKGROUND: Oxathiapiprolin is a novel fungicide and the first of the piperidinyl-thiazole-isoxazoline class to be discovered. This fungicide has been reported to have high activity against Plasmopara viticola, the grapevine downy mildew agent, and other plant-pathogenic oomycetes. In this study, the baseline sensitivity of Italian P. viticola populations towards oxathiapiprolin was established on 29 samples collected in 10 different viticultural areas. Two insensitive strains were characterized for their mechanism of resistance. RESULTS: Oxathiapiprolin exhibited substantial inhibitory activity against 27 of the 29 populations tested, with EC50 values ranging from a minimum of under 4 × 10-5  mg L-1 to over 4 × 10-1  mg L-1 , with an average value of 3.2 × 10-2  mg L-1 . Two stable suspected oxathiapiprolin-resistant mutants were isolated from population exhibiting reduced sensitivity, and sequenced for the oxathiapiprolin target gene PvORP1. The comparison with wild-type isolates revealed that the resistant isolates possessed a heterozygous mutation causing the amino acid substitution N837I, recently reported in the literature. CONCLUSION: The results obtained indicate a risk for Italian P. viticola populations to develop resistance to oxathiapiprolin in association with the N837I mutation at PvORP1. Anti-resistance strategies should be carefully implemented and the sensitivity levels to this molecule should be monitored accurately in future to preserve its effectiveness. © 2022 Society of Chemical Industry.

The Study of the Germination Dynamics of Plasmopara viticola Oospores Highlights the Presence of Phenotypic Synchrony With the Host.

The plant disease onset is a complex event that occurs when the pathogen and the host encounter in a favorable environment. While the plant-pathogen interaction has been much investigated, little attention has been given to the phenological synchrony of the event, especially when both plant and pathogen overwinter, as in the case of grapevines and the downy mildew agent, the oomycete Plasmopara viticola. Oospores allow this obligate parasite to survive grapevine dormancy and, germinating, produce inoculum for primary infections. During overwintering, environmental factors influence the potential oospore germination. This study aimed at investigating the existence of synchrony between the pathogen and the host by identifying and quantifying the most important factors determining oospore maturation and germination and the relationship existing with grapevine phenology. Generalized linear models (GLM and GLMM) were used to analyze the germination dynamics of the oospores overwintered in controlled and field conditions and incubated in isothermal conditions, and oospore viability tests were carried out at different time points. Results showed that the most indicative parameter to describe the germination dynamics is the time spent by the oospores from the start of overwintering. The oospores overwintered in field showed phenological traits related to grapevine phenology not observed in controlled conditions. In particular, they completed the maturation period by the end of grapevine dormancy and germinated more rapidly at plant sprouting, when grapevine reaches susceptibility. Overall, the oospores proved to be able to modulate their behavior in close relationship with grapevine, showing a great adaptation to the host's phenology.

Fungicide Resistance Evolution and Detection in Plant Pathogens: Plasmopara viticola as a Case Study.

The use of single-site fungicides to control plant pathogens in the agroecosystem can be associated with an increased selection of resistance. The evolution of resistance represents one of the biggest challenges in disease control. In vineyards, frequent applications of fungicides are carried out every season for multiple years. The agronomic risk of developing fungicide resistance is, therefore, high. Plasmopara viticola, the causal agent of grapevine downy mildew, is a high risk pathogen associated with the development of fungicide resistance. P. viticola has developed resistance to most of the fungicide classes used and constitutes one of the most important threats for grapevine production. The goals of this review are to describe fungicide resistance evolution in P. viticola populations and how to conduct proper monitoring activities. Different methods have been developed for phenotyping and genotyping P. viticola for fungicide resistance and the different phases of resistance evolution and life cycles of the pathogen are discussed, to provide a full monitoring toolkit to limit the spread of resistance. A detailed revision of the available tools will help in shaping and harmonizing the monitoring activities between countries and organizations.

Characterization of Lysinibacillus fusiformis strain S4C11: In vitro, in planta, and in silico analyses reveal a plant-beneficial microbe.

Despite sharing many of the traits that have allowed the genus Bacillus to gain recognition for its agricultural relevance, the genus Lysinibacillus is not as well-known and studied. The present study employs in vitro, in vivo, in planta, and in silico approaches to characterize Lysinibacillus fusiformis strain S4C11, isolated from the roots of an apple tree in northern Italy. The in vitro and in vivo assays demonstrated that strain S4C11 possesses an antifungal activity against different fungal pathogens, and is capable of interfering with the germination of Botrytis cinerea conidia, as well as of inhibiting its growth through the production of volatile organic molecules. In planta assays showed that the strain possesses the ability to promote plant growth, that is not host-specific, both in controlled conditions and in a commercial nursery. Biocontrol assays carried out against phytopathogenic viruses gave contrasting results, suggesting that the strain does not activate the host's defense pathways. The in silico analyses were carried out by sequencing the genome of the strain through an innovative approach that combines Illumina and High-Definition Mapping methods, allowing the reconstruction of a main chromosome and two plasmids from strain S4C11. The analysis of the genes encoded by the genome contributed to the characterization of the strain, detecting genes related to the biocontrol effect detected in the experimental trials.

Georgian Grapevine Cultivars: Ancient Biodiversity for Future Viticulture.

Grapevine (Vitis vinifera) is one of the most widely cultivated plant species of agricultural interest, and is extensively appreciated for its fruits and the wines made from its fruits. Considering the high socio-economic impact of the wine sector all over the world, in recent years, there has been an increase in work aiming to investigate the biodiversity of grapevine germplasm available for breeding programs. Various studies have shed light on the genetic diversity characterizing the germplasm from the cradle of V. vinifera domestication in Georgia (South Caucasus). Georgian germplasm is placed in a distinct cluster from the European one and possesses a rich diversity for many different traits, including eno-carpological and phenological traits; resistance to pathogens, such as oomycetes and phytoplasmas; resistance to abiotic stresses, such as sunburn. The aim of this review is to assess the potential of Georgian cultivars as a source of useful traits for breeding programs. The unique genetic and phenotypic aspects of Georgian germplasm were unraveled, to better understand the diversity and quality of the genetic resources available to viticulturists, as valuable resources for the coming climate change scenario.

Characterization of fungicide sensitivity profiles of Botrytis cinerea populations sampled in Lombardy (Northern Italy) and implications for resistance management.

BACKGROUND: Resistance to fungicides is one of the aspects that must be considered when planning treatments to achieve an optimal control of grey mold, caused by Botrytis cinerea, in vineyards. In this study, extensive fungicide resistance monitoring was carried out in Northern Italy (Lombardy region) to evaluate several aspects of fungicide resistance (frequency of resistance, effect of field treatments, mechanism of resistance and fitness) on 720 B. cinerea strains isolated from 36 vineyards. RESULTS: Of the characterized strains, 12% were resistant to a single fungicide class (3% to the succinate dehydrogenase inhibitor boscalid, 4% to the anilinopirimidine cyprodinil; 5% to the phenylpirrole fludioxonil; 0.1% to the ketoreductase inhibitor fenhexamid) and 0.8% to two fungicide classes contemporaneously. Resistance was associated with mutations reported in the literature for boscalid (H272Y/R) and fenhexamid (P238S or I232M). Two new mutations in sdhC (A187F) and in sdhD (I189L) could be related to boscalid resistance. Strains resistant to fludioxonil did not show any known mutations. No significant differences were found in the fitness of sensitive and resistant strains. CONCLUSION: Overall, field populations of B. cinerea showed a relatively low frequency of resistance, but the geographical distribution of resistance, genetic mechanisms of resistance and fitness of resistant strains suggest that management of resistance should be implemented, at local and regional levels. Particular attention should be given to the fungicide sprays planned before veraison, since they seem to be associated with a higher frequency of resistant strains in vineyards. © 2020 Society of Chemical Industry.

A time-course investigation of resistance to the carboxylic acid amide mandipropamid in field populations of Plasmopara viticola treated with anti-resistance strategies.

BACKGROUND: Despite anti-resistance strategies being recommended to reduce selection pressure on insensitive strains, no information is available on fungal population dynamics following their application in real field conditions. In this study, the effects on Plasmopara viticola populations of two identical spray programs, differing only in including or not the carboxylic acid amide (CAA) mandipropamid in mixture and in alternation with an anti-resistance partner, were compared in terms of downy mildew control efficacy and mandipropamid sensitivity in two commercial vineyards for four seasons. RESULTS: Both programs effectively and similarly protected grapevine from downy mildew, despite different starting sensitivity levels of the P. viticola populations. In the vineyard where resistant strains were initially present, the frequency of mutations associated with resistance (G1105S/V) fluctuated within seasons in both programs and a shift towards sensitivity occurred after 3 years of the mandipropamid-free program. Where sensitivity was initially present, no changes occurred in the mandipropamid-free program and resistant strains were selected in the mandipropamid program in high disease pressure conditions. CONCLUSION: The anti-resistance strategy including mandipropamid in mixture showed a good field performance, but did not completely prevent an increase in the frequency of insensitive strains. This supports the need for appropriate planning to determine which mixtures should be used in the field. © 2018 Society of Chemical Industry.

Assessment of QoI resistance in Plasmopara viticola oospores.

QoI fungicides, inhibitors of mitochondrial respiration at the Qo site of cytochrome b in the mitochondrial bc(1) enzyme complex, are commonly applied in vineyards against Plasmopara viticola (Berk. & MA Curtis) Berl. & De Toni. Numerous treatments per year with QoI fungicides can lead to the selection of resistant strains in the pathogen population owing to the very specific and efficient mode of action. In order to evaluate the resistance risk and its development, two different methods, biological and molecular, were applied to measure the sensitivity of oospores differentiated in vineyards, both treated and untreated with azoxystrobin, from 2000 to 2004. Assays using oospores have the advantage of analysing the sensitivity of bulked samples randomly collected in vineyards, describing accurately the status of resistance at the end of the grapevine growing season. Both methods correlated well in describing the resistance situation in vineyards. QoI resistance was not observed in one vineyard never treated with QoI fungicides. In the vineyard where azoxystrobin had been used in mixture with folpet, the selection of QoI-resistant strains was lower, compared with using solely QoI. In vineyards where QoI treatments have been stopped, a decrease in resistance was generally observed.

Assessing pigmented pericarp of maize kernels as possible source of resistance to fusarium ear rot, Fusarium spp. infection and fumonisin accumulation.

One of the purposes of maize genetic improvement is the research of genotypes resistant to fusarium ear rot (FER) and fumonisin accumulation. Flavonoids in the pericarp of the kernels are considered particularly able to reduce the fumonisin accumulation (FUM). The aim of this field study was to assess the effect of flavonoids, associated with anti-insect protection and Fusarium verticillioides inoculation, on FER symptoms and fumonisin contamination in maize kernels. Two isogenic hybrids, one having pigmentation in the pericarp (P1-rr) and the other without it (P1-wr), were compared. P1-rr showed lower values of FER symptoms and FUM contamination than P1-wr only if the anti-insect protection and the F. verticillioides inoculations were applied in combination. Fusarium spp. kernel infection was not influenced by the presence of flavonoids in the pericarp. Artificial F. verticillioides inoculation was more effective than anti-insect protection in enhancing the inhibition activity of flavonoids toward FUM contamination. The interactions between FUM contamination levels and FER ratings were better modeled in the pigmented hybrid than in the unpigmented one. The variable role that the pigment played in kernel defense against FER and FUM indicates that flavonoids alone may not be completely effective in the resistance of fumonisin contamination in maize.

Sensitivity to cymoxanil in Italian populations of Plasmopara viticola oospores.

BACKGROUND: The level of sensibility towards cymoxanil was quantified by oospore germination assays in 28 populations of Plasmopara viticola (Berk. et Curt.) Berlese and De Toni sampled from different Italian regions from 2009 to 2012. RESULTS: The populations showed good sensitivity levels, with EC50 values often lower than 10 mg AI L(-1) and percentages of resistant individuals lower than 16%. Only three populations, sampled at the end of the 2012 grapevine growing season, were characterised by high resistance levels. Field trials carried out in two of these vineyards showed that, at the beginning of the 2013 grapevine growing season, the EC50 values of P. viticola populations as measured in the sporangial assay were higher than those observed with oospores. At the end of the season, in plots where cymoxanil was not applied, the populations fully reverted to sensitivity, while the EC50 values remained high where 3-6 applications were performed. CONCLUSION: Oospore germination assays provide valuable information on the sensitivity of populations in vineyards also at the quantitative level. The results obtained during the grapevine growing season confirm those obtained on the oospores, and that cymoxanil resistance is unstable, indirectly suggesting that the application of the fungicide according to antiresistance strategies can lead to good disease control.

Mating behavior of a Northern Italian population of Fusarium verticillioides associated with maize.

Fusarium verticillioides, the most common causal organism of Fusarium stalk and ear rot of maize in Northern Italy, produces important mycotoxins such as fumonisins. Reproductive biology of F. verticillioides has been widely studied in numerous maize growing areas, but up to now no information is available on the mating behavior and genetic structure of this plant pathogen in Italy. Mating type and female fertility distribution and effective population number, N ( e ), were assessed for a population of 181 F. verticillioides strains isolated from three fields located in Lombardia region (Northern Italy) during 2007-2008 maize growing season. The ratio of MAT-1:MAT-2 was significantly different from the theoretical 1:1 ratio expected in an idealized population in which individuals mate at random. The frequency of hermaphroditic strains was 20 % of the total population. N ( e ) for mating type was 89 % of the count (total population) and the N ( e ) for male or hermaphrodite status was 55 %. The number of isolates that can function as the female parent limited N ( e ) in the examined population. Under equilibrium cycle, assuming that female fertility has been lost due to selection and mutation rate during asexual reproduction, sexual reproduction needed to occur only once per 40 to 118 asexual generations to maintain this level of sexual fertility.