A comparative cell wall analysis of Trichoderma spp. confirms a conserved polysaccharide scaffold and suggests an important role for chitosan in mycoparasitism.

Fungal cell walls are dynamic extracellular matrices that enable efficient adaptation to changing environments. While the cell wall compositions of yeasts, human, and plant pathogenic fungi have been studied to some extent, the cell walls of mycoparasites remain poorly characterized. Trichoderma species comprise a diverse group of soil fungi with different survival strategies and lifestyles. The comparative study of cell wall carbohydrate-active enzymes in 13 Trichoderma spp. revealed that the types of enzymes involved in chitin and chitosan metabolism are phylogenetically distant between mycoparasitic and saprotrophic species. Here, we compare the carbohydrate composition and function of the cell wall of a saprotrophic strain Trichoderma reesei with that of the mycoparasitic, biological control agent Trichoderma atroviride. Monosaccharide and glycosidic linkage analyses as well as dual in situ interaction assays showed that the cell wall polysaccharide composition is conserved between both species, except for the amounts of chitin detected. The results suggest that the observed accumulation of chitosan during mycoparasitism may prevent host recognition. Remarkably, Trichoderma atroviride undergoes dynamic cell wall adaptations during both vegetative development and mycoparasitism, which appears to be confirmed by an evolutionarily expanded group of specialized enzymes. Overall, our analyses support the notion that habitat specialization is reflected in cell wall architecture and that plastic chitin remodeling may confer an advantage to mycoparasites, ultimately enabling the successful invasion and parasitism of plant pathogens. This information may potentially be exploited for the control of crop diseases using biological agents. IMPORTANCE: Trichoderma species are emerging model fungi for the development of biocontrol agents and are used in industrial biotechnology as efficient enzyme producers. Fungal cell walls are complex structures that differ in carbohydrate, protein, and enzyme composition across taxa. Here, we present a chemical characterization of the cell walls of two Trichoderma spp., namely the predominantly saprotrophic Trichoderma reesei and the mycoparasite Trichoderma atroviride. Chemical profiling revealed that Trichoderma spp. remodel their cell wall to adapt to particular lifestyles, with dynamic changes during vegetative development. Importantly, we found that chitosan accumulation during mycoparasitism of a fungal host emerged as a sophisticated strategy underpinning an effective attack. These insights shed light on the molecular mechanisms that allow mycoparasites to overcome host defenses and can be exploited to improve the application of T. atroviride in biological pest control. Moreover, our results provide valuable information for targeting the fungal cell wall for therapeutic purposes.

Are Basic Substances a Key to Sustainable Pest and Disease Management in Agriculture? An Open Field Perspective.

Pathogens and pests constantly challenge food security and safety worldwide. The use of plant protection products to manage them raises concerns related to human health, the environment, and economic costs. Basic substances are active, non-toxic compounds that are not predominantly used as plant protection products but hold potential in crop protection. Basic substances' attention is rising due to their safety and cost-effectiveness. However, data on their protection levels in crop protection strategies are lacking. In this review, we critically analyzed the literature concerning the field application of known and potential basic substances for managing diseases and pests, investigating their efficacy and potential integration into plant protection programs. Case studies related to grapevine, potato, and fruit protection from pre- and post-harvest diseases and pests were considered. In specific cases, basic substances and chitosan in particular, could complement or even substitute plant protection products, either chemicals or biologicals, but their efficacy varied greatly according to various factors, including the origin of the substance, the crop, the pathogen or pest, and the timing and method of application. Therefore, a careful evaluation of the field application is needed to promote the successful use of basic substances in sustainable pest management strategies in specific contexts.

Sequence, structure and functionality of pectin methylesterases and their use in sustainable carbohydrate bioproducts: A review.

Pectin methylesterases (PMEs) are enzymes that play a critical role in modifying pectins, a class of complex polysaccharides in plant cell walls. These enzymes catalyze the removal of methyl ester groups from pectins, resulting in a change in the degree of esterification and consequently, the physicochemical properties of the polymers. PMEs are found in various plant tissues and organs, and their activity is tightly regulated in response to developmental and environmental factors. In addition to the biochemical modification of pectins, PMEs have been implicated in various biological processes, including fruit ripening, defense against pathogens, and cell wall remodelling. This review presents updated information on PMEs, including their sources, sequences and structural diversity, biochemical properties and function in plant development. The article also explores the mechanism of PME action and the factors influencing enzyme activity. In addition, the review highlights the potential applications of PMEs in various industrial sectors related to biomass exploitation, food, and textile industries, with a focus on development of bioproducts based on eco-friendly and efficient industrial processes.

Methods for Fungicide Efficacy Screenings: Multiwell Testing Procedures for the Oomycetes Phytophthora infestans and Pythium ultimum.

Oomycetes-borne diseases represent a serious problem for agriculture sustainability due to the high use of chemical products employed for their control. In recent years, increasing concerns on side effects associated with fungicide utilization have led to the reduction of the permissible modes of action, with the remaining ones continuously threatened by the increase of resistant strains in the pathogen populations. In this context, it is mandatory to develop new generation fungicides characterized by high specificity towards the target species and low environmental impact to guarantee the sustainability, productivity, and quality of food production. Fungicide discovery is a lengthy and costly process, and despite these urgent needs, poor description and formalization of high-throughput methodologies for screening the efficacy of active compounds are commonly reported for these kinds of organisms. In this study, a comprehensive picture of two high-throughput practices for efficient fungicide screening against plant-pathogenic oomycetes has been provided. Different protocols using multiwell plates were validated on approved crop protection products using Phytophthora infestans and Pythium ultimum as the model species. In addition, detailed statistical inputs useful for the analysis of data related to the efficacy of screenings are included.

Influence of Nitrogen on Grapevine Susceptibility to Downy Mildew.

Downy mildew, caused by the obligate parasite Plasmopara viticola, is one of the most important threats to viticulture. The exploitation of resistant and susceptibility traits of grapevine is one of the most promising ways to increase the sustainability of disease management. Nitrogen (N) fertilization is known for influencing disease severity in the open field, but no information is available on its effect on plant-pathogen interaction. A previous RNAseq study showed that several genes of N metabolism are differentially regulated in grapevine upon P. viticola inoculation, and could be involved in susceptibility or resistance to the pathogen. The aim of this study was to evaluate if N fertilization influences: (i) the foliar leaf content and photosynthetic activity of the plant, (ii) P. viticola infectivity, and (iii) the expression of the candidate susceptibility/resistance genes. Results showed that N level positively correlated with P. viticola infectivity, confirming that particular attention should be taken in vineyard to the fertilization, but did not influence the expression of the candidate genes. Therefore, these genes are manipulated by the pathogen and can be exploited for developing new, environmentally friendly disease management tools, such as dsRNAs, to silence the susceptibility genes or breeding for resistance.

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.

Pathogen Adaptation to American (Rpv3-1) and Eurasian (Rpv29) Grapevine Loci Conferring Resistance to Downy Mildew.

Durable resistance is a key objective in genetic improvement for disease resistance in grapevines, which must survive for years in the field in the presence of adaptable pathogen populations. In this study, the adaptation of 72 Northern Italian isolates of Plasmopara viticola, the downy mildew agent, has been investigated into Bianca, possessing Rpv3-1, the most frequently exploited resistance locus for genetic improvement, and Mgaloblishvili, a Vitis vinifera variety possessing the newly discovered Rpv29 locus. Infection parameters (latency period, infection frequency, and disease severity) and oospore production and viability were evaluated and compared to those of Pinot noir, the susceptible reference. The expected levels of disease control were achieved by both resistant cultivars (>90% on Bianca; >25% on Mgaloblishvili), despite the high frequency of isolates able to grow on one (28%) or both (46%) accessions. The disease incidence and severity were limited by both resistant cultivars and the strains able to grow on resistant accessions showed signatures of fitness penalties (reduced virulence, infection frequency, and oospore density). Together, these results indicate an adequate pathogen control but suitable practices must be adopted in the field to prevent the diffusion of the partially adapted P. viticola strains to protect resistance genes from erosion.

RNAi of a Putative Grapevine Susceptibility Gene as a Possible Downy Mildew Control Strategy.

Downy mildew, caused by the oomycete Plasmopara viticola, is one of the diseases causing the most severe economic losses to grapevine (Vitis vinifera) production. To date, the application of fungicides is the most efficient method to control the pathogen and the implementation of novel and sustainable disease control methods is a major challenge. RNA interference (RNAi) represents a novel biotechnological tool with a great potential for controlling fungal pathogens. Recently, a candidate susceptibility gene (VviLBDIf7) to downy mildew has been identified in V. vinifera. In this work, the efficacy of RNAi triggered by exogenous double-stranded RNA (dsRNA) in controlling P. viticola infections has been assessed in a highly susceptible grapevine cultivar (Pinot noir) by knocking down VviLBDIf7 gene. The effects of dsRNA treatment on this target gene were assessed by evaluating gene expression, disease severity, and development of vegetative and reproductive structures of P. viticola in the leaf tissues. Furthermore, the effects of dsRNA treatment on off-target (EF1α, GAPDH, PEPC, and PEPCK) and jasmonic acid metabolism (COI1) genes have been evaluated. Exogenous application of dsRNA led to significant reductions both in VviLBDIf7 gene expression, 5 days after the treatment, and in the disease severity when artificial inoculation was carried out 7 days after dsRNA treatments. The pathogen showed clear alterations to both vegetative (hyphae and haustoria) and reproductive structures (sporangiophores) that resulted in stunted growth and reduced sporulation. Treatment with dsRNA showed signatures of systemic activity and no deleterious off-target effects. These results demonstrated the potential of RNAi for silencing susceptibility factors in grapevine as a sustainable strategy for pathogen control, underlying the possibility to adopt this promising biotechnological tool in disease management strategies.

From plant resistance response to the discovery of antimicrobial compounds: The role of volatile organic compounds (VOCs) in grapevine downy mildew infection.

The discovery of new mechanisms of resistance and natural bioactive molecules could be two of the possible ways to reduce fungicide use in vineyard and assure an acceptable and sustainable protection against Plasmopara viticola, the grapevine downy mildew agent. Emission of volatile organic compounds (VOCs), such as terpenes, norisoprenoids, alcohols and aldehydes, is frequently induced in plants in response to attack by pathogens, such as P. viticola, that is known to cause a VOCs increment in cultivars harboring American resistance traits. In this study, the role of leaf VOCs in the resistance mechanism of two resistant cultivars (Mgaloblishvili, a pure Vitis vinifera cultivar, and Bianca, an interspecific hybrid) and the direct antimicrobial activity of four selected VOCs have been investigated. The leaf VOCs profiles, analyzed through solid-phase microextraction gas chromatography-mass spectrometry analysis, as well as the expression of six terpene synthases (TPSs), were determined upon pathogen inoculation. In both cultivars, the expression pattern of six TPSs increased soon after pathogen inoculation and an increment of nine VOCs has been detected. While in Mgaloblishvili VOCs were synthesized early after P. viticola inoculation, they constituted a late response to pathogen in Bianca. All the four terpenes (farnesene, nerolidol, ocimene and valencene), chosen according to the VOC profiles and gene expression analysis, caused a significant reduction (53-100%) in P. viticola sporulation. These results support the role of VOCs into defense mechanisms of both cultivars and suggest their potential role as a natural and eco-friendly solution to protect grapevine from P. viticola.

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.