Engineering d‐limonene synthase down‐regulation in orange fruit induces resistance against the fungus Phyllosticta citricarpa through enhanced accumulation of monoterpene alcohols and activation of defence

Terpene volatiles play an important role in the interactions between specialized pathogens and fruits. Citrus black spot (CBS), caused by the fungus Phyllosticta citricarpa, is associated with crop losses in different citrus‐growing areas worldwide. The pathogen may infect the fruit for 20–24 weeks after petal fall, but the typical hard spot symptoms appear when the fruit have almost reached maturity, caused by fungal colonization and the induction of cell lysis around essential oil cavities. d ‐Limonene represents approximately 95% of the total oil gland content in mature orange fruit. Herein, we investigated whether orange fruit with reduced d ‐limonene content in peel oil glands via an antisense (AS) approach may affect fruit interaction with P. citricarpa relative to empty vector (EV) controls. AS fruit showed enhanced resistance to the fungus relative to EV fruit. Because of the reduced d ‐limonene content, an over‐accumulation of linalool and other monoterpene alcohols was found in AS relative to EV fruit. A global gene expression analysis at 2 h and 8 days after inoculation with P. citricarpa revealed the activation of defence responses in AS fruit via the up‐regulation of different pathogenesis‐related (PR) protein genes, probably as a result of enhanced constitutive accumulation of linalool and other alcohols. When assayed in vitro and in vivo, monoterpene alcohols at the concentrations present in AS fruit showed strong antifungal activity. We show here that terpene engineering in fruit peels could be a promising method for the development of new strategies to obtain resistance to fruit diseases.     

Characterization of protein kinase PsSRPKL,a novel pathogenicity factor in the wheat stripe rust fungus

As in other eukaryotes, protein kinases (PKs) are generally evolutionarily conserved and play major regulatory roles in plant pathogenic fungi. Many PKs have been proven to be important for pathogenesis in model fungal plant pathogens, but little is currently known about their roles in the pathogenesis of cereal rust fungi, devastating pathogens in agriculture worldwide. Here, we report on an in planta highly induced PK gene PsSRPKL from the wheat stripe rust fungus Puccinia striiformis f. sp. tritici (Pst), one of the most important cereal rust fungi. PsSRPKL belongs to a group of PKs that are evolutionarily specific to cereal rust fungi. It shows a high level of intraspecies polymorphism in the kinase domains and directed green fluorescent protein chimers to plant nuclei. Overexpression of PsSRPKL in fission yeast induces aberrant cell morphology and a decreased resistance to environmental stresses. Most importantly, PsSRPKL is proven to be an important pathogenicity factor responsible for fungal growth and responses to environmental stresses, therefore contributing significantly to Pst virulence in wheat. We hypothesize that cereal rust fungi have developed specific PKs as pathogenicity factors for adaptation to their host species during evolution. Thus, our findings provide significant insights into pathogenicity and virulence evolution in cereal rust fungi.     

Antimicrobial volatile organic compounds affect morphogenesis-related enzymes in Guignardia citricarpa,causal agent of citrus black spot

Although non-volatile substances toxic to plant pathogenic microorganisms have been extensively studied over the years, few studies have focused on microbial volatile organic compounds (VOCs). The VOCs produced by the yeast Saccharomyces cerevisiae strain CR-1, used in fermentative processes for fuel ethanol production, are able to inhibit the vegetative development of the fungus Guignardia citricarpa, causal agent of the disease citrus black spot. How microbial VOCs affect the development of fungi is not known. Thus, the objective of the present work was to study the effect of the artificial mixture of VOCs identified from S. cerevisiae on intracellular enzymes involved in the mycelial morphogenesis in G. citricarpa. The phytopathogenic fungus was exposed to artificial mixture of VOCs constituted by alcohols (ethanol, 3-methyl-1-butanol, 2-methyl-1-butanol and phenylethyl alcohol) and esters (ethyl acetate and ethyl octanoate) in the proportions naturally found in the atmosphere produced by the yeast. The VOCs inhibited considerably the mycelial development and interfered negatively with the production of the morphogenesis-related enzymes. After 72 h of exposure to the VOCs the laccase and tyrosinase activities decreased 46 and 32%, respectively, however, the effect on the chitinase and β-1,3-glucanase activities was lower, 17 and 13% of inhibition, respectively. Therefore, the exposure of the fungus to the antimicrobial volatiles can influence both fungal mycelial growth rate and activity of enzymes implicated in morphogenesis. This knowledge is important to understand the microbial interactions mediated by VOCs in nature and to develop new strategies to control plant pathogens as G. citricarpa in postharvest.     

A TaqMan PCR Method for Routine Diagnosis of the Quarantine Fungus Guignardia citricarpa on Citrus Fruit

With respect to disease risk for the quarantine fungus Guignardia citricarpa on citrus fruit an accurate diagnosis for routine analysis is required. Also, when inspections have to be performed on imported citrus fruits, a fast detection method is urgently needed. A fast automated DNA extraction method based on magnetic beads combined with a real‐time PCR assay was optimized to improve and advance the routine diagnosis of citrus black spot disease. Real‐time PCR was used for detection of the pathogen G. citricarpa in planta. A specific primer/TaqMan probe combination that discriminates between G. citricarpa and the harmless citrus endophyte Guignardia mangiferae, was designed based on the internal transcribed spacer region of the multi‐copy rDNA gene. Co‐amplification of target DNA along with an internal competitor DNA fragment made the diagnostic assay more reliable to check for false negatives. The real‐time PCR was specific, since no cross reaction was observed with a series of citrus pathogens and related species. The diagnostic assay was performed on lesions dissected from imported diseased oranges. Comparison between the conventional PCR and the real‐time PCR methods showed that the TaqMan method was more sensitive.     

Volatile organic compounds produced by Saccharomyces cerevisiae inhibit the in vitro development of Guignardia citricarpa,the causal agent of citrus black spot

Due to the low chemical control effectiveness of citrus black spot, caused by the fungus Guignardia citricarpa at postharvest, and to the search for alternative control methods, this study aimed to evaluate the in vitro effect of volatile organic compounds (VOCs), produced by yeast Saccharomyces cerevisiae, on G. citricarpa. It was observed that the yeast strains evaluated acted as antagonists by VOC production, whose maximum inhibitory capacity was as high as 87.2%. The presence of fermentable carbon sources in the medium was essential for the bioactive VOC production by the yeast. The analysis of VOCs produced in PDA medium by SPME–GC–MS indicated the presence of high quantities of alcohols as well as esters. An artificial VOC mixture prepared on the basis of the composition of the VOCs mimicked the inhibitory effects of the natural VOCs released by S. cerevisiae. Thus, the VOCs produced by the yeast or the artificial mixtures can be a promising control method for citrus black spot or others postharvest diseases.     

Identification of the New Pathogen (Stemphylium lycopersici) Causing Leaf Spot on Pepino (Solanum muricatum)

Pepino (Solanum muricatum var. pepino) plants were found affected by an extensive leaf spot caused by plant pathogenic fungi during a survey in the Cameron highlands, Pahang state, Malaysia. Symptomatic leaf samples were collected from infected pepino plants and cultivated on PDA medium, and the pathogen was isolated and purified; then, consequently, all isolates were identified as Stemphylium lycopersici on the basis of their cultural and morphological characteristics and combined sequences of the internal transcribed spacer (ITS) and glyceraldehyde‐3‐phosphate dehydrogenase (gpd) regions. A pathogenicity assay on detached leaves further confirmed that S. lycopersici causes leaf spot disease. To the best of our knowledge, this is the first report of S. lycopersici causing leaf spot on pepino in Malaysia and worldwide.     

Culture storage age and fungal re‐isolation from host‐tissue influence Colletotrichum spp. virulence to pepper fruits

Using resistant cultivars is the most sustainable and practical approach against plant diseases. Plant germplasm and breeding lines are selected and assayed against, usually, the most aggressive or virulent strains of a pathogen (e.g., fungus) that causes the disease. However, prolong storage of the pathogen in culture media could affect virulence that, consequently, also influence the outcome of the resistance assay. This study demonstrates that long‐term storage (at least a year) of Colletotrichum truncatum and C. scovillei, causal agents of pepper anthracnose, in potato dextrose agar (PDA) medium decreased the aggressiveness and virulence of the fungus in host‐pepper fruits. However, reintroduction of the pathogen to the host and isolation of the pathogen as the new inoculum, prior to inoculation assays, increased the virulence of the fungi. These findings suggest that re‐inoculation and re‐isolation of Colletotichum truncatum and C. scovillei that have been stored for at least 1 year in PDA medium are necessary when using fungal cultures in pathogenicity and plant resistance assays to achieve desirable, comparable and reliable results.     

Agrobacterium-mediated transformation of Guignardia citricarpa: An efficient tool to gene transfer and random mutagenesis

Guignardia citricarpa is the causal agent of Citrus Black Spot (CBS), an important disease in Citriculture. Due to the expressive value of this activity worldwide, especially in Brazil, understanding more about the functioning of this fungus is of utmost relevance, making possible the elucidation of its infection mechanisms, and providing tools to control CBS. This work describes for the first time an efficient and successful methodology for genetic transformation of G. citricarpa mycelia, which generated transformants expressing the gene encoding for the gfp (green fluorescent protein) and also their interaction with citrus plant. Mycelia of G. citricarpa were transformed via Agrobacterium tumefaciens, which carried the plasmid pFAT-gfp, contains the genes for hygromycin resistance (hph) as well as gfp. The optimization of the agrotransformation protocol was performed testing different conditions (type of membrane; inductor agent concentration [acetosyringone – AS] and cocultivation time). Results demonstrated that the best condition occurred with the utilization of cellulose's ester membrane; 200 μM of AS and 96 h as cocultivation time. High mitotic stability (82 %) was displayed by transformants using Polymerase Chain Reaction (PCR) technique to confirm the hph gene insertion. In addition, the presence of gfp was observed inside mycelia by epifluorescence optical microscopy. This technique easy visualization of the behaviour of the pathogen interacting with the plant for the first time, allowing future studies on the pathogenesis of this fungus. The establishment of a transformation method for G. citricarpa opens a range of possibilities and facilitates the study of insertional mutagenesis and genetic knockouts, in order to identify the most important genes involved in the pathogenesis mechanisms and plant–pathogen interaction.     

Melanization of appressoria is critical for the pathogenicity of <Emphasis Type="Italic">Diplocarpon rosae</Emphasis>

Previous studies have shown the role of melanized appressoria in the pathogenicity of various fungi. Diplocarpon rosae is a worldwide outdoor fungal pathogen of rose plants causing black spot disease of rose leaves. To fully understand how this fungus colonizes its host, which is critical for the development of an efficient and sustainable disease management program, we studied the fungal (especially the appressoria) structures of D. rosae in detail at an early stage of infection. Using both microscopic and biochemical analyses, we observed strong melanized appressoria formation localized at the point of D. rosae penetration, which forms the pathogen–plant interface. Treatment of infected plants with melanin biosynthesis inhibitors (MBIs) prevented melanization of D. rosae appressoria and positively correlated with significant reductions in black spot disease symptoms, suggesting that melanization of appressoria might be a critical factor for the pathogenicity of D. rosae. Our findings were confirmed and validated by the lack of melanized appressorial ring formation on an artificial surface and on a D. rosae-non host plant system, Arabidopsis thaliana. Our findings suggest that localized melanization of appressoria is a crucial factor for the pathogenicity of D. rosae and treatment of the fungus with MBIs seems to be a promising disease management alternative for black spot disease of roses.     

Suppression of Rust and Brown Eye Spot Diseases on Coffee by Phosphites and By‐products of Coffee and Citrus Industries

The rust and brown eye spot, caused by Hemileia vastatrix and Cercospora coffeicola, respectively, are the most important fungal diseases on coffee in South America. Their management is mainly by chemical treatment, and there is no genetic resistance to brown eye spot known so far. Considering the need for developing alternative products for their control, the goal of this work was to evaluate the effects of phosphites and by‐products of coffee and citrus industries on rust and brown eye spot. Formulations of coffee and citrus industry by‐products, phosphites and their combination with fungicide were evaluated in field experiments, and their effect on fungal urediniospores and conidia was evaluated in vitro. In the field, treatments were applied individually or in combination and the in vitro assays were performed with manganese phosphite (Reforce Mn), potassium phosphite and citrus industry by‐product (Fortaleza), copper phosphite and coffee industry by‐product (Fitoforce Full), and fungicide. The severity and incidence of rust and brown eye spot on coffee leaves, yield, and leaf retention were evaluated in the field. Percentage of spore germination was evaluated in vitro for both fungi, whereas mycelial growth was evaluated for C. coffeicola only. The treatments Fortaleza, Reforce Mn and Fitoforce Full suppressed both diseases with a reduction in defoliation. In the year 2012, the plants treated with Reforce Mn and Reforce Mn + Fortaleza showed a yield increase of 72 and 88%, respectively, which was similar to the results shown by the fungicide treatment. In vitro inhibition of germination of H. vastatrix urediniospores and of C. coffeicola conidia was observed and suggests that the products exert some toxic effects to both fungi. Finally, the results observed indicate that the combined use of by‐products of plant‐processing industries and phosphites is an alternative and can be added efficiently to the management of coffee diseases.     

Secretome Analysis Identifies Potential Pathogenicity/Virulence Factors of Tilletia indica,a Quarantined Fungal Pathogen Inciting Karnal Bunt Disease in Wheat

Tilletia indica is a smut fungus that incites Karnal bunt in wheat. It has been considered as quarantine pest in more than 70 countries. Despite its quarantine significance, there is meager knowledge regarding the molecular mechanisms of disease pathogenesis. Moreover, various disease management strategies have proven futile. Development of effective disease management strategy requires identification of pathogenicity / virulence factors. With this aim, the present study was conducted to compare the secretomes of T. indica isolates, that is, highly (TiK) and low (TiP) virulent isolates. About 120 and 95 protein spots were detected reproducibly in TiK and TiP secretome gel images. Nineteen protein spots, which were consistently observed as upregulated/differential in the secretome of TiK isolate, were selected for their identification by MALDI‐TOF/TOF. Identified proteins exhibited homology with fungal proteins playing important role in fungal adhesion, penetration, invasion, protection against host‐derived reactive oxygen species, production of virulence factors, cellular signaling, and degradation of host cell wall proteins and antifungal proteins. These results were complemented with T. indica genome sequence leading to identification of candidate pathogenicity / virulence factors homologs that were further subjected to sequence‐ and structure‐based functional annotation. Thus, present study reports the first comparative secretome analysis of T. indica for identification of pathogenicity / virulence factors. This would provide insights into pathogenic mechanisms of T. indica and aid in devising effective disease management strategies.     

Comparative proteomic analysis of Xanthomonas citri ssp. citri periplasmic proteins reveals changes in cellular envelope metabolism during in vitro pathogenicity induction

Citrus canker is a plant disease caused by Gram‐negative bacteria from the genus Xanthomonas. The most virulent species is Xanthomonas citri ssp. citri (XAC), which attacks a wide range of citrus hosts. Differential proteomic analysis of the periplasm‐enriched fraction was performed for XAC cells grown in pathogenicity‐inducing (XAM‐M) and pathogenicity‐non‐inducing (nutrient broth) media using two‐dimensional electrophoresis combined with liquid chromatography‐tandem mass spectrometry. Amongst the 40 proteins identified, transglycosylase was detected in a highly abundant spot in XAC cells grown under inducing condition. Additional up‐regulated proteins related to cellular envelope metabolism included glucose‐1‐phosphate thymidylyltransferase, dTDP‐4‐dehydrorhamnose‐3,5‐epimerase and peptidyl‐prolyl cis–trans‐isomerase. Phosphoglucomutase and superoxide dismutase proteins, known to be involved in pathogenicity in other Xanthomonas species or organisms, were also detected. Western blot and quantitative real‐time polymerase chain reaction analyses for transglycosylase and superoxide dismutase confirmed that these proteins were up‐regulated under inducing condition, consistent with the proteomic results. Multiple spots for the 60‐kDa chaperonin and glyceraldehyde‐3‐phosphate dehydrogenase were identified, suggesting the presence of post‐translational modifications. We propose that substantial alterations in cellular envelope metabolism occur during the XAC infectious process, which are related to several aspects, from defence against reactive oxygen species to exopolysaccharide synthesis. Our results provide new candidates for virulence‐related proteins, whose abundance correlates with the induction of pathogenicity and virulence genes, such as hrpD6, hrpG, hrpB7, hpa1 and hrpX. The results present new potential targets against XAC to be investigated in further functional studies.     

Contribution of a harpin protein from Xanthomonas axonopodis pv. citri to pathogen virulence

Xanthomonas axonopodis pv. citri (Xac), the bacterium that causes citrus canker, contains a gene in the hrp [for hypersensitive response (HR) and pathogenicity] cluster that encodes a harpin protein called Hpa1. Hpa1 produced HR in the nonhost plants tobacco, pepper and Arabidopsis, whereas, in the host plant citrus, it elicited a weak defence response with no visible phenotype. Co‐infiltrations of Xac with or without the recombinant Hpa1 protein in citrus leaves produced a larger number of cankers in the presence of the protein. To characterize the effect of Hpa1 during the disease, an XacΔhpa1 mutant was constructed, and infiltration of this mutant caused a smaller number of cankers. In addition, the lack of Hpa1 hindered bacterial aggregation both in solution and in planta. Analysis of citrus leaves infiltrated with Hpa1 revealed alterations in mesophyll morphology caused by the presence of cavitations and crystal idioblasts, suggesting the binding of the harpin to plant membranes and the elicitation of signalling cascades. Overall, these results suggest that, even though Hpa1 elicits the defence response in nonhost plants and, to a lesser extent, in host plants, its main roles in citrus canker are to alter leaf mesophyll structure and to aggregate bacterial cells, and thus increase virulence and pathogen fitness. We expressed the N‐terminal and C‐terminal regions and found that, although both regions elicited HR in nonhost plants, only the N‐terminal region showed increased virulence and bacterial aggregation, supporting the role of this region of the protein as the main active domain.     

An RXLR effector secreted by Phytophthora parasitica is a virulence factor and triggers cell death in various plants

RXLR effectors encoded by Phytophthora species play a central role in pathogen–plant interactions. An understanding of the biological functions of RXLR effectors is conducive to the illumination of the pathogenic mechanisms and the development of disease control strategies. However, the virulence function of Phytophthora parasitica RXLR effectors is poorly understood. Here, we describe the identification of a P. parasitica RXLR effector gene, PPTG00121 (PpE4), which is highly transcribed during the early stages of infection. Live cell imaging of P. parasitica transformants expressing a full-length PpE4 (E4FL)-mCherry protein indicated that PpE4 is secreted and accumulates around haustoria during plant infection. Silencing of PpE4 in P. parasitica resulted in significantly reduced virulence on Nicotiana benthamiana. Transient expression of PpE4 in N. benthamiana in turn restored the pathogenicity of the PpE4-silenced lines. Furthermore, the expression of PpE4 in both N. benthamiana and Arabidopsis thaliana consistently enhanced plant susceptibility to P. parasitica. These results indicate that PpE4 contributes to pathogen infection. Finally, heterologous expression experiments showed that PpE4 triggers non-specific cell death in a variety of plants, including tobacco, tomato, potato and A. thaliana. Virus-induced gene silencing assays revealed that PpE4-induced cell death is dependent on HSP90, NPK and SGT1, suggesting that PpE4 is recognized by the plant immune system. In conclusion, PpE4 is an important virulence RXLR effector of P. parasitica and recognized by a wide range of host plants.     

Functional genomics tools to decipher the pathogenicity mechanisms of the necrotrophic fungus Plectosphaerella cucumerina in Arabidopsis thaliana

The analysis of the interaction between Arabidopsis thaliana and adapted (PcBMM) and nonadapted (Pc2127) isolates of the necrotrophic fungus Plectosphaerella cucumerina has contributed to the identification of molecular mechanisms controlling plant resistance to necrotrophs. To characterize the pathogenicity bases of the virulence of necrotrophic fungi in Arabidopsis, we developed P. cucumerina functional genomics tools using Agrobacterium tumefaciens‐mediated transformation. We generated PcBMM‐GFP and Pc2127‐GFP transformants constitutively expressing the green fluorescence protein (GFP), and a collection of random T‐DNA insertional PcBMM transformants. Confocal microscopy analyses of the initial stages of PcBMM‐GFP infection revealed that this pathogen, like other necrotrophic fungi, does not form an appressorium or penetrate into plant cells, but causes successive degradation of leaf cell layers. By comparing the colonization of Arabidopsis wild‐type plants and hypersusceptible (agb1‐1 and cyp79B2cyp79B3) and resistant (irx1‐6) mutants by PcBMM‐GFP or Pc2127‐GFP, we found that the plant immune response was already mounted at 12–18 h post‐inoculation, and that Arabidopsis resistance to these fungi correlated with the time course of spore germination and hyphal growth on the leaf surface. The virulence of a subset of the PcBMM T‐DNA insertional transformants was determined in Arabidopsis wild‐type plants and agb1‐1 mutant, and several transformants were identified that showed altered virulence in these genotypes in comparison with that of untransformed PcBMM. The T‐DNA flanking regions in these fungal mutants were successfully sequenced, further supporting the utility of these functional genomics tools in the molecular characterization of the pathogenicity of necrotrophic fungi.