Effects of Exogenous Application of Methyl Jasmonate and Salicylic Acid on the Physiological and Molecular Response of 'Dusa' Avocado to Rosellinia necatrix.

Methyl jasmonate (MeJA) and salicylic acid (SA) are important in mediating plant responses to abiotic and biotic stresses. MeJA and SA can act as elicitors by triggering plant defense responses similar to those induced by pathogens and may even provide long-term protection against them. Thus, exogenous application of MeJA and SA could protect susceptible avocado plants against white root rot (WRR) disease caused by the necrotrophic fungus Rosellinia necatrix, one of the main diseases affecting avocado orchards. This work evaluates the effects of MeJA or SA on the physiological and molecular response of susceptible 'Dusa' avocado rootstock and their ability to provide some protection against WRR. The application of MeJA and SA in avocado increased photoprotective mechanisms (nonphotochemical chlorophyll fluorescence quenching) and upregulated the glutathione S-transferase, suggesting the triggering of mechanisms closely related to oxidative stress relief and reactive oxygen species scavenging. In contrast to SA, MeJA's effects were more pronounced at the morphoanatomical level, including functional traits such as high leaf mass area, high stomatal density, and high root/shoot ratio, closely related to strategies to cope with water scarcity and WRR disease. Moreover, MeJA upregulated a greater number of defense-related genes than SA, including a glu protease inhibitor, a key gene in avocado defense against R. necatrix. The overall effects of MeJA increased 'Dusa' avocado tolerance to R. necatrix by inducing a primed state that delayed WRR disease symptoms. These findings point toward the use of MeJA application as an environmentally friendly strategy to mitigate the impact of this disease on susceptible avocado orchards.

Rosellinia necatrix infection induces differential gene expression between tolerant and susceptible avocado rootstocks.

Rosellinia necatrix is the causal agent of avocado white root rot (WRR). Control of this soil-borne disease is difficult, and the use of tolerant rootstocks may present an effective method to lessen its impact. To date, no studies on the molecular mechanisms regulating the avocado plant response towards this pathogen have been undertaken. To shed light on the mechanisms underpinning disease susceptibility and tolerance, molecular analysis of the gene's response in two avocado rootstocks with a contrasting disease reaction was assessed. Gene expression profiles against R. necatrix were carried out in the susceptible 'Dusa' and the tolerant selection BG83 avocado genotypes by micro-array analysis. In 'Dusa', the early response was mainly related to redox processes and cell-wall degradation activities, all becoming enhanced after disease progression affected photosynthetic capacity, whereas tolerance to R. necatrix in BG83 relied on the induction of protease inhibitors and their negative regulators, as well as genes related to tolerance to salt and osmotic stress such as aspartic peptidase domain-containing proteins and gdsl esterase lipase proteins. In addition, three protease inhibitors were identified, glu protease, trypsin and endopeptidase inhibitors, which were highly overexpressed in the tolerant genotype when compared to susceptible 'Dusa', after infection with R. necatrix, reaching fold change values of 52, 19 and 38, respectively. The contrasting results between 'Dusa' and BG83 provide new insights into the different mechanisms involved in avocado tolerance to Phytophthora cinnamomi and R. necatrix, which are consistent with their biotrophic and necrotrophic lifestyles, respectively. The differential induction of genes involved in salt and osmotic stress in BG83 could indicate that R. necatrix penetration into the roots is associated with osmotic effects, suggesting that BG83's tolerance to R. necatrix is related to the ability to withstand osmotic imbalance. In addition, the high expression of protease inhibitors in tolerant BG83 compared to susceptible 'Dusa' after infection with the pathogen suggests the important role that these proteins may play in the defence of avocado rootstocks against R. necatrix.

Response of the Biocontrol Agent Pseudomonas pseudoalcaligenes AVO110 to Rosellinia necatrix Exudate.

The rhizobacterium Pseudomonas pseudoalcaligenes AVO110, isolated by the enrichment of competitive avocado root tip colonizers, controls avocado white root rot disease caused by Rosellinia necatrix Here, we applied signature-tagged mutagenesis (STM) during the growth and survival of AVO110 in fungal exudate-containing medium with the goal of identifying the molecular mechanisms linked to the interaction of this bacterium with R. necatrix A total of 26 STM mutants outcompeted by the parental strain in fungal exudate, but not in rich medium, were selected and named growth-attenuated mutants (GAMs). Twenty-one genes were identified as being required for this bacterial-fungal interaction, including membrane transporters, transcriptional regulators, and genes related to the metabolism of hydrocarbons, amino acids, fatty acids, and aromatic compounds. The bacterial traits identified here that are involved in the colonization of fungal hyphae include proteins involved in membrane maintenance (a dynamin-like protein and ColS) or cyclic-di-GMP signaling and chemotaxis. In addition, genes encoding a DNA helicase (recB) and a regulator of alginate production (algQ) were identified as being required for efficient colonization of the avocado rhizosphere.IMPORTANCE Diseases associated with fungal root invasion cause a significant loss of fruit tree production worldwide. The bacterium Pseudomonas pseudoalcaligenes AVO110 controls avocado white root rot disease caused by Rosellinia necatrix by using mechanisms involving competition for nutrients and niches. Here, a functional genomics approach was conducted to identify the bacterial traits involved in the interaction with this fungal pathogen. Our results contribute to a better understanding of the multitrophic interactions established among bacterial biocontrol agents, the plant rhizosphere, and the mycelia of soilborne pathogens.

Usage of the Heterologous Expression of the Antimicrobial Gene afp From Aspergillus giganteus for Increasing Fungal Resistance in Olive.

The antifungal protein (AFP) produced by Aspergillus giganteus, encoded by the afp gene, has been used to confer resistance against a broad range of fungal pathogens in several crops. In this research, transgenic olive plants expressing the afp gene under the control of the constitutive promoter CaMV35S were generated and their disease response against two root infecting fungal pathogens, Verticillium dahliae and Rosellinia necatrix, was evaluated. Embryogenic cultures derived from a mature zygotic embryo of cv. 'Picual' were used for A. tumefaciens transformation. Five independent transgenic lines were obtained, showing a variable level of afp expression in leaves and roots. None of these transgenic lines showed enhanced resistance to Verticillium wilt. However, some of the lines displayed a degree of incomplete resistance to white root rot caused by R. necatrix compared with disease reaction of non-transformed plants or transgenic plants expressing only the GUS gene. The level of resistance to this pathogen correlated with that of the afp expression in root and leaves. Our results indicate that the afp gene can be useful for enhanced partial resistance to R. necatrix in olive, but this gene does not protect against V. dahliae.

Interactions between the Powdery Mildew Effector BEC1054 and Barley Proteins Identify Candidate Host Targets.

There are over 500 candidate secreted effector proteins (CSEPs) or Blumeria effector candidates (BECs) specific to the barley powdery mildew pathogen Blumeria graminis f.sp. hordei. The CSEP/BEC proteins are expressed and predicted to be secreted by biotrophic feeding structures called haustoria. Eight BECs are required for the formation of functional haustoria. These include the RNase-like effector BEC1054 (synonym CSEP0064). In order to identify host proteins targeted by BEC1054, recombinant BEC1054 was expressed in E. coli, solubilized, and used in pull-down assays from barley protein extracts. Many putative interactors were identified by LC-MS/MS after subtraction of unspecific binders in negative controls. Therefore, a directed yeast-2-hybrid assay, developed to measure the effectiveness of the interactions in yeast, was used to validate putative interactors. We conclude that BEC1054 may target several host proteins, including a glutathione-S-transferase, a malate dehydrogenase, and a pathogen-related-5 protein isoform, indicating a possible role for BEC1054 in compromising well-known key players of defense and response to pathogens. In addition, BEC1054 interacts with an elongation factor 1 gamma. This study already suggests that BEC1054 plays a central role in barley powdery mildew virulence by acting at several levels.

Expression of the ß-1,3-glucanase gene bgn13.1 from Trichoderma harzianum in strawberry increases tolerance to crown rot diseases but interferes with plant growth.

The expression of antifungal genes from Trichoderma harzianum, mainly chitinases, has been used to confer plant resistance to fungal diseases. However, the biotechnological potential of glucanase genes from Trichoderma has been scarcely assessed. In this research, transgenic strawberry plants expressing the ß-1,3-glucanase gene bgn13.1 from T. harzianum, under the control of the CaMV35S promoter, have been generated. After acclimatization, five out of 12 independent lines analysed showed a stunted phenotype when growing in the greenhouse. Moreover, most of the lines displayed a reduced yield due to both a reduction in the number of fruit per plant and a lower fruit size. Several transgenic lines showing higher glucanase activity in leaves than control plants were selected for pathogenicity tests. When inoculated with Colletotrichum acutatum, one of the most important strawberry pathogens, transgenic lines showed lower anthracnose symptoms in leaf and crown than control. In the three lines selected, the percentage of plants showing anthracnose symptoms in crown decreased from 61 % to a mean value of 16.5 %, in control and transgenic lines, respectively. Some transgenic lines also showed an enhanced resistance to Rosellinia necatrix, a soil-borne pathogen causing root and crown rot in strawberry. These results indicate that bgn13.1 from T. harzianum can be used to increase strawberry tolerance to crown rot diseases, although its constitutive expression affects plant growth and fruit yield. Alternative strategies such as the use of tissue specific promoters might avoid the negative effects of bgn13.1 expression in plant performance.

Broadly Conserved Fungal Effector BEC1019 Suppresses Host Cell Death and Enhances Pathogen Virulence in Powdery Mildew of Barley (Hordeum vulgare L.).

The interaction of barley, Hordeum vulgare L., with the powdery mildew fungus Blumeria graminis f. sp. hordei is a well-developed model to investigate resistance and susceptibility to obligate biotrophic pathogens. The 130-Mb Blumeria genome encodes approximately 540 predicted effectors that are hypothesized to suppress or induce host processes to promote colonization. Blumeria effector candidate (BEC)1019, a single-copy gene encoding a putative, secreted metalloprotease, is expressed in haustorial feeding structures, and host-induced gene silencing of BEC1019 restricts haustorial development in compatible interactions. Here, we show that Barley stripe mosaic virus-induced gene silencing of BEC1019 significantly reduces fungal colonization of barley epidermal cells, demonstrating that BEC1019 plays a central role in virulence. In addition, delivery of BEC1019 to the host cytoplasm via Xanthomonas type III secretion suppresses cultivar nonspecific hypersensitive reaction (HR) induced by Xanthomonas oryzae pv. oryzicola, as well as cultivar-specific HR induced by AvrPphB from Pseudomonas syringae pv. phaseolicola. BEC1019 homologs are present in 96 of 241 sequenced fungal genomes, including plant pathogens, human pathogens, and free-living nonpathogens. Comparative analysis revealed variation at several amino acid positions that correlate with fungal lifestyle and several highly conserved, noncorrelated motifs. Site-directed mutagenesis of one of these, ETVIC, compromises the HR-suppressing activity of BEC1019. We postulate that BEC1019 represents an ancient, broadly important fungal protein family, members of which have evolved to function as effectors in plant and animal hosts.

Host-induced gene silencing in barley powdery mildew reveals a class of ribonuclease-like effectors.

Obligate biotrophic pathogens of plants must circumvent or counteract defenses to guarantee accommodation inside the host. To do so, they secrete a variety of effectors that regulate host immunity and facilitate the establishment of pathogen feeding structures called haustoria. The barley powdery mildew fungus Blumeria graminis f. sp. hordei produces a large number of proteins predicted to be secreted from haustoria. Fifty of these Blumeria effector candidates (BEC) were screened by host-induced gene silencing (HIGS), and eight were identified that contribute to infection. One shows similarity to ß-1,3 glucosyltransferases, one to metallo-proteases, and two to microbial secreted ribonucleases; the remainder have no similarity to proteins of known function. Transcript abundance of all eight BEC increases dramatically in the early stages of infection and establishment of haustoria, consistent with a role in that process. Complementation analysis using silencing-insensitive synthetic cDNAs demonstrated that the ribonuclease-like BEC 1011 and 1054 are bona fide effectors that function within the plant cell. BEC1011 specifically interferes with pathogen-induced host cell death. Both are part of a gene superfamily unique to the powdery mildew fungi. Structural modeling was consistent, with BEC1054 adopting a ribonuclease-like fold, a scaffold not previously associated with effector function.

Developing tools to unravel the biological secrets of Rosellinia necatrix, an emergent threat to woody crops.

UNLABELLED: White root rot caused by Rosellinia necatrix is one of the most destructive diseases of many woody plants in the temperate regions of the world, particularly in Europe and Asia. Recent outbreaks of R. necatrix around the globe have increased the interest in this pathogen. Although the ecology of the disease has been poorly studied, recent genetic and molecular advances have opened the way for future detailed studies of this fungus. TAXONOMY: Rosellinia necatrix Prilleux. Kingdom Fungi; subdivision Ascomycotina; class Euascomycetes; subclass Pyrenomycetes; order Sphaeriales, syn. Xylariales; family Xylariaceae; genus Rosellinia. IDENTIFICATION: Fungal mycelium is present on root surfaces and under the bark, forming mycelium fans, strands or cords. A typical presence of pear-shaped or pyriform swellings can be found above the hyphal septum (with diameters of up to 13 µm). Sclerotia are black, hard and spherical nodules, several millimetres in diameter. Black sclerotia crusts may also form on roots. On synthetic media, it forms microsclerotia: irregular rough bodies composed of a compact mass of melanized, interwoven hyphae with no differentiated cells. Chlamydospores are almost spherical (15 µm in diameter). Synnemata, also named coremia (0.5-1.5 mm in length), can be formed from sclerotia or from mycelial masses. Conidia (3-5 µm in length and 2.5-3 µm in width) are very difficult to germinate in vitro. Ascospores are monostichous, situated inside a cylindrical, long-stalked ascus. They are ellipsoidal and cymbiform (36-46 µm in length and 5.5-6.3 µm in width). HOST RANGE: This fungus can attack above 170 different plant hosts from 63 genera and 30 different families, including vascular plants and algae. Some are of significant economic importance, such as Coffea spp., Malus spp., Olea europaea L., Persea americana Mill., Prunus spp. and Vitis vinifera L. DISEASE SYMPTOMS: Rosellinia necatrix causes white (or Dematophora) root rot, which, by aerial symptoms, shows a progressive weakening of the plant, accompanied by a decline in vigour. The leaves wilt and dry, and the tree can eventually die. White cottony mycelium and mycelial strands can be observed in the crown and on the root surface. On woody plant roots, the fungus can be located between the bark and the wood, developing typical mycelium fans, invading the whole root and causing general rotting. DISEASE CONTROL: Some approaches have been attempted involving the use of tolerant plants and physical control (solarization). Chemical control in the field and biological control methods are still under development.

Two similar enhanced root-colonizing Pseudomonas strains differ largely in their colonization strategies of avocado roots and Rosellinia necatrix hyphae.

Pseudomonas alcaligenes AVO73 and Pseudomonas pseudoalcaligenes AVO110 were selected previously as efficient avocado root tip colonizers, displaying in vitro antagonism towards Rosellinia necatrix, causal agent of avocado white root rot. Despite the higher number of antagonistic properties shown in vitro by AVO73, only AVO110 demonstrated significant protection against avocado white root rot. As both strains are enhanced root colonizers, and as colonization is crucial for the most likely biocontrol mechanisms used by these strains, namely production of non-antibiotic antifungal compounds and competition for nutrients and niches, we decided to compare the interactions of the bacterial strains with avocado roots as well as with R. necatrix hyphae. The results indicate that strain AVO110 is superior in biocontrol trait swimming motility and establishes on the root tip of avocado plants faster than AVO73. Visualization studies, using Gfp-labelled derivatives of these strains, showed that AVO110, in contrast to AVO73, colonizes intercellular crevices between neighbouring plant root epidermal cells, a microhabitat of enhanced exudation. Moreover, AVO110, but not AVO73, also colonizes root wounds, described to be preferential penetration sites for R. necatrix infection. This result strongly suggests that AVO110 meets, and can attack, the pathogen on the root. Finally, when co-inoculated with the pathogen, AVO110 utilizes hyphal exudates more efficiently for proliferation than AVO73 does, and colonizes the hyphae more abundantly than AVO73. We conclude that the differences between the strains in colonization levels and strategies are likely to contribute to, and even can explain, the difference in disease-controlling abilities between the strains. This is the first report that shows that two similar bacterial strains, selected by their ability to colonize avocado root, use strongly different root colonization strategies and suggests that in addition to the total bacterial root colonization level, the sites occupied on the root are important for biocontrol.

Selection for biocontrol bacteria antagonistic toward Rosellinia necatrix by enrichment of competitive avocado root tip colonizers.

Biological control of soil-borne pathogens is frequently based on the application of antagonistic microorganisms selected solely for their ability to produce in vitro antifungal factors. The aim of this work was to select bacteria that efficiently colonize the roots of avocado plants and display antagonism towards Rosellinia necatrix, the causal agent of avocado white root rot. A high frequency of antagonistic strains (ten isolates, 24.4%) was obtained using a novel procedure based on the selection of competitive avocado root tip colonizers. Amplification and sequencing of the 16S rRNA gene, in combination with biochemical characterization, showed that eight and two of the selected isolates belonged to the genera Pseudomonas and Stenotrophomonas, respectively. Characterization of antifungal compounds produced by the antagonistic strains showed variable production of exoenzymes and HCN. Only one of these strains, Pseudomonas sp. AVO94, produced a compound that could be related to antifungal antibiotics. All of the ten selected strains showed twitching motility, a cell movement involved in competitive colonization of root tips. Production of N-acyl-homoserine lactones and indole-3-acetic acid was also reported for some of these isolates. Resistance to several bacterial antibiotics was tested, and three strains showing resistance to only one of them were selected for biocontrol assays. The three selected strains persisted in the rhizosphere of avocado plants at levels considered crucial for efficient biocontrol, 10(5)-10(6) colony forming units/g of root; two of them, Pseudomonas putida AVO102 and Pseudomonas pseudoalcaligenes AVO110, demonstrated significant protection of avocado plants against white root rot.