Factors affecting the Trichoderma harzianum-induced resistance against downy mildew of grapevine and interaction between Plasmopara viticola genotypes co-inoculated in the host

Grapevine (Vitis vinifera L.) is one of the major fruit crops worldwide and varieties used for table grape or wine production are susceptible to several pathogens. Downy mildew caused by the oomycete Plasmopara viticola is an important grapevine disease that threatens leaves and young berries and, to avoid yield losses, control of the disease is based on the application of chemical fungicides. Genetic analysis of pathogen’s population structure in field indicated that germination of oospores, causing primary infections occurs for a long period, alongside secondary infection cycles, and few P. viticola genotypes are dominating during an epidemic. Alternative methods for controlling downy mildew have been studied, including the use of microbial biocontrol agents. Application of Trichoderma harzianum T39 (T39) has been shown to reduce downy mildew symptoms in grapevine by activating the plant mediated resistance mechanism. Induced resistance offers the prospect of broad-spectrum disease control using the plant’s own defenses and represents a promising low-impact tool for controlling crop diseases. However, the potential of induced resistance has yet to be fully realized, mainly due to its only partial control of the disease and its inconsistency under field conditions. Induced resistance is a plant-mediated mechanism, and its expression under field conditions is likely to be influenced by a number of factors, including environment, genotype and crop nutrition. Concerns about the impact of abiotic stresses on agriculture have been raised in the last decade, especially in light of the predicted effects of climate changes. High temperatures and drought associated with heat waves may occur with increased frequency as a result of climate change, threatening crop production and influencing interactions with both pathogenic and beneficial microorganisms. Aim of this project was to study the downy mildew disease form the pathogen and from the host plant point of views, in order to maximize the control of the disease with low-impact natural methods. To evaluate the efficacy of the T39-induced resistance under non-optimal conditions and to study pathogen’s infection dynamics of different P. viticola isolates we evaluated i) the T39-induced resistance in plants exposed to heat and drought stresses, ii) the T39-induced resistance in different grapevine cultivars and iii) the possible selection mechanisms of different P. viticola isolates. The work was structured in three distinct sections where, at first, we could assess that co-inoculated P. viticola isolates competed for the infection of the host, although being equally infective when singularly inoculated. Competition was not related to the origin of the isolate and we hypothesized that competitive selection was modulated by differences in the 2 secretion of effector molecules, which explained the establishment of dominant genotypes over an epidemic season. In other two sections we demonstrated that T39-induced resistance was found to be reduced in plants exposed to the combination of heat and drought stresses, moreover, variable levels of efficacy were observed in different grapevine cultivars. Modulation of the marker genes in the T39-induced resistance was partially attenuated in plants under heat and drought stress. The molecular mechanisms activated in response to the resistance inducer were different and complex among cultivars, indicating that specific receptors are probably involved in the regulation of the plant response. The work presented in this thesis provides a deeper understanding to current knowledge of the biology of this grapevine pathogen and of the mechanisms of the induced resistance. Different P. viticola genotypes reacted differently when co-existing on a unique substrate, indicating that plant-pathogen interactions seem to be more complex than mere colonization of the plant tissue. The environmental conditions and the plant genotype are key factors affecting the T39-induced resistance. Therefore, prevention of predominant genotypes during an epidemic season and consideration of the variable responsiveness of the plant to the resistance inducer applied are important strategies for the improvement of biocontrol methods against downy mildew of grapevin

Characterization of Trichoderma harzianum T39 induced resistance against Plasmopara viticola during abiotic stresses

Climate change will increase the temperature and decrease precipitations in several areas of the world, increasing heat and drought stress for plants. Abiotic stress response in plants is largely controlled by the hormone ABA while the defence against biotic stresses is controlled by salicylic acid (SA) and jasmonic acid (JA)/ethylene signalling pathways. ABA signalling plays a crucial role in the modulation of defence reactions under multiple stress exposure. Thus, environmental stress is an additional factor to consider when studying the plant defence responses. Downy mildew of grapevine, (Plasmopara viticola) is one of the most destructive diseases and plants are treated with chemical fungicides to avoid substantial yield losses. To reduce the use of chemicals, a strong interest was recently focused on enhancement of plant defence. After treatment with a resistance inducer, plants react with a faster and/or stronger defence against pathogens. This defence mechanism is called induced systemic resistance (ISR). ISR protects from a broad spectrum of pathogens but is often inconsistent and likely to be influenced by the environment. Since abiotic stresses may strongly influence the induced resistance, the aim of this project was to study the effect of a heat and/or drought exposure of grapevine plants on the resistance induced by Trichoderma harzianum T39 (T39) against downy mildew. Grapevine plants were maintained at different temperatures and irrigation regimes to simulate heat and drought stress for 14 days. Soil moisture was daily measured in each pot with a soil moisture probe and leaf water potential was measured using the Sholander pressure bomb. Treatments with T39 and water (control) were applied on days 12, 13 and 14. Plants were inoculated with a P. viticola suspension (1×105 sporangia/mL) and disease severity was evaluated one week after inoculation. Leaves were sampled before and after pathogen inoculation and ISR marker genes were analysed in real time RT-PCR. The exposure of plants to heat and drought stress given singularly did not affect the efficacy of the T39 treatment, while efficacy was negatively affected when the stresses were combined. The induction of grapevine ISR marker genes were attenuated in heat and drought stressed plants. The weaker efficacy of the T39-induced resistance in heat + drought stressed plants could be related to the attenuated induction of the defence-related genes. Our results indicate that ISR could be less effective in areas where climate change will take place and when plants are exposed to heat and drought stresses. Future climate change should, therefore, be taken into account in disease management and in evaluating the efficacy of resistance inducers

Co-inoculated Plasmopara viticola genotypes compete for the infection of the host independently from the aggressiveness components

During Plasmopara viticola epidemics only few genotypes produce most of the secondary lesions and dominate in the population. Selection of dominant genotypes is hypothesized to be linked to environmental conditions and can occur rapidly, particularly if there is also difference between genotypes in terms of fitness and aggressiveness. Measurements of aggressiveness components can largely determine the rate of epidemic development, although the components of aggressiveness do not take into account potential direct competition between genotypes. Differences in aggressiveness have been also reported to be greater under non-optimal conditions suggesting for genotype adaptation to different conditions. To evaluate differences in latency at non-optimal conditions, we characterized genotypes deriving from different climatic regions at three different temperatures (15, 25 and 35 A degrees C) and we found no differences. To investigate whether other factors may impact on competition between P. viticola genotypes, we evaluated polycyclic infections of P. viticola by co-inoculating three genotypes with similar aggressiveness components in two different co-inoculation experiments and an increasing prevalence of one of the two genotypes was observed. Competition was not related to the origin of the genotype and we hypothesize that competitive selection is modulated by differences in the secretion of effector molecules which can contribute to the establishment of dominant genotypes over an epidemic seaso

Dissecting positive or negative effects of abiotic stress on grapevine self-protection induced by Trichoderma harzianum T39

Downy mildew is one of the most destructive grapevine diseases, and its control is based on the application of fungicides. There is increasing interest to reduce reliance on pesticides for disease control in viticulture and to focus on alternative methods, such as biocontrol agents. Several Trichoderma spp. strains are active against numerous plant pathogens, and their biocontrol activity is based on different mechanisms. In grapevine, T. harzianum T39 activates a plant-mediated resistance and reduces downy mildew symptoms, but its efficacy under abiotic stress conditions have not jet investigated. Our aim is to investigate the relationship between drought stress and P. viticola infection, and to characterize the efficacy of T. harzianum T39-induced resistance under water stress condition

Characterization of Trichoderma harzianum T39 induced resistance against Plasmopara viticola in heat and drought stressed grapevine plants

Plants can acquire an enhanced defensive capacity after treatment with a resistance-inducing agent that results in a faster and/or stronger defence reaction against pathogens. These agents induce a broad spectrum and long lasting resistance, but rarely provide complete control of infections (for the most part between 20 to 85% of disease control). Under field conditions, the expression of induced resistance is often inconsistent and likely to be influenced by the environment, plant genotype and its physiology. However little information is available on the role of these factors on the level of induced resistance. Since abiotic stresses may strongly influence the host plant, the aim of this project is to investigate the effect of a short heat and/or drought exposure of grapevine plants on the systemic resistance activated by Trichoderma harzianum T39 (T39) or benzothiaziadole (BTH) against downy mildew (Plasmopara viticola). In susceptible grapevine cultivars grown under controlled greenhouse conditions, foliar treatments with T39 or BTH reduce disease severity. Gene expression analysis indicates the activation of jasmonic acid and ethylene signal in the defence induced by T39, while salicylic acid pathway is activated by BTH. The pre-exposure of plants to drought and/or heat stress (abiotic stress) did not affect the efficacy of BTH-activated resistance. On the contrary, the T39-induced resistance was influenced by the plant pre-exposure to the above mentioned abiotic stress conditions, showing different levels of downy mildew reduction. The results indicate that T39-induced resistance might be a useful tool in the control of downy mildew, but, even if its efficacy level is not affect by the genotype, it can be modulated by the physiological state of the plant. Further characterization of molecular events of T39-induced resistance under abiotic stress exposure could be particularly important in order to predict the possible effect and stability of T39-induced resistance under field condition especially in a view of climate change

Abiotic stresses affect Trichoderma harzianum T39-induced resistance to downy mildew in grapevine

Enhancement of plant defense through application resistance inducers seems a promising alternative to chemical fungicides for controlling crop diseases, but the efficacy can be affected by abiotic factors in the field. Plants respond to abiotic stresses with hormonal signals that may interfere with the mechanisms of induced systemic resistance (ISR) to pathogens. In this study we exposed grapevines to heat and/or drought to investigate the effects of abiotic stresses on grapevine resistance induced by Trichoderma harzianum T39 (T39) to downy mildew. Whereas the efficacy of T39-induced resistance was not affected by exposure to heat or drought, it was significantly reduced by combined abiotic stresses. Decrease of leaf water potential and up-regulation of heat-stress markers confirmed that plants reacted to abiotic stresses. Basal expression of defense-related genes and their up-regulation during T39-induced resistance were attenuated by abiotic stresses, in agreement with the reduced efficacy of T39. The evidence reported here suggests that exposure of crops to abiotic stress should be carefully considered to optimize the use of resistance inducers, especially in view of future global climate changes. Expression analysis of ISR markers genes could be helpful to identify when plants are responding to abiotic stresses, in order to optimize treatments with resistance inducers in fiel

Relevance of the plant genotype for biocontrol tools based on resistance induction

Downy mildew caused by Plasmopara viticola is a serious disease of grapevine, and its control is based on the intense application of chemical fungicide. We previously shown Trichoderma harzianum T39 (T39) and benzothiadiazole-7-carbothioic acid S-methyl ester (BTH) reduce downy mildew severity in the Pinot Noir grapevine variety by enhancing plant resistance. However, the effect of the plant genotype on the resistance induction mechanisms was not yet analyzed in grapevine. The aim of this study was to characterize the physiological and molecular properties of the T39-induced resistance in different grapevine varieties used for table and wine grape production, in order to further optimize the use of this agent for downy mildew control. Rooted cutting from 14 grapevine varieties were grown under greenhouse conditions. T39 conidia, BTH and water (control) were applied to grapevine leaves and Plasmopara viticola sporangia were then inoculated. Here we showed that T39 treatment significantly reduces downy mildew symptoms in the different grapevine varieties. However, different levels of T39 efficacy were observed: T39 efficacy was particularly high in Negroamaro plants, but it was lower in Primitivo than in Pinot Noir plants, indicating that the plant genotype is a key determinant of T39-induced resistance. Moreover, the efficacy of resistance activated by BTH was higher than T39 and was not affected by the grapevine genotype. Gene expression analysis of four defence marker genes confirms the complex effect of the plant genotype on the molecular mechanism activated by pathogen inoculation and by T39 treatment in different varieties. In Pinot Noir and Primitivo varieties, T39-induced resistance is based on a dual effect: direct induction of PR-2 and PR-4 genes and enhanced expression of these genes after pathogen inoculation. Correlation between gene expression and T39 efficacy was observed in Primitivo and Pinot Noir plants, but more complex regulation or additional defence genes are implicated in the T39-induced resistance in the variety Sugraone and Negroamaro. Grapevine varieties have different reaction to the same stimuli, indicating that specific receptors are probably involved in the regulation of the plant response. The evidence reported here suggests that a good resistance inducer should be applied on a highly responsive cultivar in order to maximize the effect of the biocontrol agent

Genomics to plant health: effect of climate change on plant self-protection

Beneficial
 microorganisms 
could
 be 
an 
effective 
approach
 to
 reduce
 the 
use 
of 
chemicals 
in 
agriculture.
 In
 the 
case‐studies 
here
 reported, the 
fungus
 Trichoderma 
harzianum
 T39 and
 the 
bacterium
 Bacillus 
amyloliquefaciens 
S499 
show
 promising 
results
 in
 inducing
 resistance 
against 
pathogen