Appressorial Formation Research Articles

MoCAP proteins regulated by MoArk1-mediated phosphorylation coordinate endocytosis and actin dynamics to govern development and virulence of Magnaporthe oryzae.

Actin organization is a conserved cellular process that regulates the growth and development of eukaryotic cells. It also governs the virulence process of pathogenic fungi, such as the rice blast fungus Magnaporthe oryzae, with mechanisms not yet fully understood. In a previous study, we found that actin-regulating kinase MoArk1 displays conserved functions important in endocytosis and actin organization, and MoArk1 is required for maintaining the growth and full virulence of M. oryzae. To understand how MoArk1 might function, we identified capping protein homologs from M. oryzae (MoCAP) that interact with MoArk1 in vivo. MoCAP is heterodimer consisting of α and β subunits MoCapA and MoCapB. Single and double deletions of MoCAP subunits resulted in abnormal mycelial growth and conidia formation. The ΔMocap mutants also exhibited reduced appressorium penetration and invasive hyphal growth within host cells. Furthermore, the ΔMocap mutants exhibited delayed endocytosis and abnormal cytoskeleton assembly. Consistent with above findings, MoCAP proteins interacted with MoAct1, co-localized with actin during mycelial development, and participated in appressorial actin ring formation. Further analysis revealed that the S85 residue of MoCapA and the S285 residue of MoCapB were subject to phosphorylation by MoArk1 that negatively regulates MoCAP functions. Finally, the addition of exogenous phosphatidylinositol 4,5-bisphosphate (PIP2) failed to modulate actin ring formation in ΔMocap mutants, in contrast to the wild-type strain, suggesting that MoCAP may also mediate phospholipid signaling in the regulation of the actin organization. These results together demonstrate that MoCAP proteins whose functions are regulated by MoArk1 and PIP2 are important for endocytosis and actin dynamics that are directly linked to growth, conidiation and pathogenicity of M. oryzae.

Multifaceted Roles of the Ras Guanine-Nucleotide Exchange Factor ChRgf in Development, Pathogenesis, and Stress Responses of Colletotrichum higginsianum

The infection process of Colletotrichum higginsianum, which causes a disease of crucifers, involves several key steps: conidial germination, appressorial formation, appressorial penetration, and invasive growth in host tissues. In this study, the ChRgf gene encoding a Ras guanine-nucleotide exchange factor protein was identified by screening T-DNA insertion mutants generated from Agrobacterium tumefaciens-mediated transformation that were unable to cause disease on the host Arabidopsis thaliana. Targeted gene deletion of ChRgf resulted in a null mutant (ΔChrgf-42) with defects in vegetative growth, hyphal morphology, and conidiation, and poor surface attachment and low germination on hydrophobic surfaces; however, there were no apparent differences in appressorial turgor pressure between the wild type and the mutant. The conidia of the mutant were unable to geminate on attached Arabidopsis leaves and did not cause any disease symptoms. Intracellular cyclic adenosine monophosphate levels in the ΔChrgf mutant were lower than that of the wild type. Our results suggest that ChRgf is a key regulator in response to salt and osmotic stresses in C. higginsianum, and indicate that it is involved in fungal pathogenicity. This gene seems to act as an important modulator upstream of several distinct signaling pathways that are involved in regulating vegetative growth, conidiation, infection-related structure development, and stress responses of C. higginsianum.

The Herbivore-Induced Plant Volatiles Methyl Salicylate and Menthol Positively affect Growth and Pathogenicity of Entomopathogenic Fungi

Some herbivore-induced-plant volatiles (HIPVs) compounds are vital for the functioning of an ecosystem, by triggering multi-trophic interactions for natural enemies, plants and herbivores. However, the effect of these chemicals, which play a crucial role in regulating the multi-trophic interactions between plant-herbivore-entomopathogenic fungi, is still unknown. To fill this scientific gap, we therefore investigated how these chemicals influence the entomopathogenic fungi growth and efficacy. In this study, Lipaphis erysimi induced Arabidopsis thaliana HIPVs were collected using headspace system and detected with GC-MS, and then analyzed the effects of these HIPVs chemicals on Lecanicillium lecanii strain V3450. We found that the HIPVs menthol and methyl salicylate at 1 and 10 nmol·ml−1 improved many performance aspects of the fungus, such as germination, sporulation, appressorial formation as well as its pathogenicity and virulence. These findings are not only important for understanding the multi-trophic interactions in an ecosystem, but also would contribute for developing new and easier procedures for conidial mass production as well as improve the pathogenicity and virulence of entomopathogenic fungi in biological pest management strategies.

Pex14/17, a filamentous fungus-specific peroxin, is required for the import of peroxisomal matrix proteins and full virulence of Magnaporthe oryzae.

Peroxisomes are ubiquitous organelles in eukaryotic cells that fulfil a variety of biochemical functions. The biogenesis of peroxisomes requires a variety of proteins, named peroxins, which are encoded by PEX genes. Pex14/17 is a putative recently identified peroxin, specifically present in filamentous fungal species. Its function in peroxisomal biogenesis is still obscure and its roles in fungal pathogenicity have not yet been documented. Here, we demonstrate the contributions of Pex14/17 in the rice blast fungus Magnaporthe oryzae (Mopex14/17) to peroxisomal biogenesis and fungal pathogenicity by targeting gene replacement strategies. Mopex14/17 has properties of both Pex14 and Pex17 with regard to its protein sequence. Mopex14/17 is distributed at the peroxisomal membrane and is essential for efficient peroxisomal targeting of proteins containing peroxisomal targeting signal 1. MoPEX19 deletion leads to the cytoplasmic distribution of Mopex14/17, indicating that the peroxisomal import of Pex14/17 is dependent on Pex19. The knockout mutants of MoPEX14/17 show reduced fatty acid utilization, reactive oxygen species (ROS) degradation and cell wall integrity. Moreover, Δmopex14/17 mutants show delayed conidial generation and appressorial formation, and a reduction in appressorial turgor accumulation and penetration ability in host plants. These defects result in a significant reduction in the virulence of the mutant. These data indicate that MoPEX14/17 plays a crucial role in peroxisome biogenesis and contributes to fungal development and pathogenicity.

Fruit maturity and post-harvest environmental conditions influence the pre-penetration stages of Monilinia infections in peaches

Brown rot caused by the fungi Monilinia laxa (Aderhold and Ruhland) Honey, M. fructicola (Winter) Honey, or M. fructigena (Aderhold and Ruhland) is a serious fungal disease of peaches. The fungal infection process begins when fungal conidia germinate on the fruit surface to produce germ tubes and/or appressoria, and the incidence of brown rot increases as fruit approaches maturity. The interaction between the fungal infection process, peach maturity, and the environmental conditions is not well understood. Accordingly, the objectives of this investigation were to investigate germ tube and appressorial formation by M. laxa and M. fructicola when they were exposed to peach skin from mature and immature fruit at various temperatures and relative humidities (RHs). The greatest number of germ tubes was found when M. laxa or M. fructicola was incubated in culture medium which contained a skin extract of mature peaches. In contrast, the greatest number of appressoria was found when M. laxa or M. fructicola was incubated in culture medium which contained a skin extract of immature peaches. Although M. fructicola produced the same number of germ tubes and appressoria at 4°C, M. fructicola produced more germ tubes than appressoria at temperatures higher than 10°C. M. laxa produced more germ tubes than appressoria at any temperature, except when it was incubated for 48h on culture medium which contained a skin extract of immature peaches at 10°C at 80% or 100% RH, or at 25°C at 60% RH. M. laxa conidia germinated better than M. fructicola conidia at low temperatures. Germ tube and appressorial formation by Monilinia spp. were influenced by fruit postharvest handling. The number of germ tubes that were formed by M. laxa conidia was significantly greater than that for M. fructicola when the conidia were incubated at 100% RH, and this number increased after 3days of refrigeration. The number of appressoria that were formed by both Monilinia spp. also increased after 3 consecutive days of refrigeration. Negligible or no germination of M. fructicola and M. laxa conidia occurred when the RH was 60%. We concluded that the dissimilar abilities of M. laxa and M. fructicola to germinate and form appressoria at low temperatures conferred a competitive advantage to M. laxa to survive during fruit postharvest refrigeration and cold storage at 4°C.

Suppression of Magnaporthe oryzae and interaction between Bacillus subtilis and rice plants in the control of rice blast

Magnaporthe oryzae, the causative pathogen of rice blast, has caused extensive losses to rice cultivation worldwide. Strains of the bacterium Bacillus subtilis have been used as biocontrol agents against rice blast. However, little has been reported about the interaction between B. subtilis and the rice plant and its mechanism of action. Here, the colonization process and induced disease resistance by B. subtilis SYX04 and SYX20 in rice plants was examined. Strains of B. subtilis labeled with green fluorescent protein reached population of more than 5 × 106 CFU/g after 20 days on mature rice leaves and were detected after 3 days on newly grown leaves. Results showed that SYX04 and SYX20 not only inhibited spore germination, germ tube length, and appressorial formation but also caused a series of alterations in the structures of hyphae and conidia. The cell walls and membrane structures of the fungus showed ultrastructural abnormalities, which became severely degraded as observed through scanning electron microscopy and transmission electron microscopy. The mixture of both B. subtilis and M. oryzae resulted in enhanced activity of peroxidase, and polyphenol oxidase while there was significantly more superoxide dismutase activity in plants that had been sprayed with B.subtilis alone. The present study suggests that colonized SYX04 and SYX20 strains protected rice plants and exhibited antifungal activity and induced systemic resistance, thus indicating their potential biological control agents.

Volatiles from Plants Induced by Multiple Aphid Attacks Promote Conidial Performance of Lecanicillium lecanii.

Herbivore-induced plant volatiles (HIPVs) are clues that help predatory insects search for food. The hypothesis that entomopathogenic fungi, which protect plants, benefit from the release of HIPVs was tested. The plant Arabidopsis thaliana was used as the source of HIPVs. The insect herbivore Lipaphis erysimi (Kaltenbach) was used as the inducer, and the fungal pathogen of the aphid Lecanicillium lecanii was exposed to HIPVs to test our hypothesis. When exposed to aphid-induced A. thaliana volatiles, the mortality of aphids pre-treated with a conidial suspension of L. lecanii, the conidial germination and the appressorial formation were significantly increased compared with the control. The decan-3-ol and 4-methylpentyl isothiocyanate that were detected in the headspace seemed to have positive and negative affection, respectively. Moreover, HIPVs generated from groups of eight aphids per plant promoted significantly increased conidial germination and appressorial formation compared with HIPVs from groups of one, two and four aphids per plant. Our results demonstrated that the pathogenicity of the entomopathogenic fungus L. lecanii was enhanced when exposed to HIPVs and that the HIPVs were affected by the number of insect herbivores that induced them.

ChSte7 Is Required for Vegetative Growth and Various Plant Infection Processes in Colletotrichum higginsianum.

Colletotrichum higginsianum is an important hemibiotrophic phytopathogen that causes crucifer anthracnose in various regions of the world. In many plant-pathogenic fungi, the Ste11-Ste7-Fus3/Kss1 kinase pathway is essential to pathogenicity and various plant infection processes. To date, the role of ChSte7 in C. higginsianum encoding a MEK orthologue of Ste7 in Saccharomyces cerevisiae has not been elucidated. In this report, we investigated the function of ChSte7 in the pathogen. The ChSte7 is predicted to encode a 522-amino-acid protein with a S_TKc conserved domain that shares 44% identity with Ste7 in S. cerevisiae. ChSte7 disruption mutants showed white colonies with irregularly shaped edges and extremely decreased growth rates and biomass productions. The ChSte7 disruption mutants did not form appressoria and showed defects in pathogenicity on leaves of Arabidopsis thaliana. When inoculated onto wounded leaf tissues, the ChSte7 disruption mutants grew only on the surface of host tissues but failed to cause lesions beyond the wound site. In contrast, both the wild-type and complementation strains showed normal morphology, produced appressoria, and caused necrosis on leaves of Arabidopsis. Analysis with qRT-PCR suggested that ChSte7 was highly expressed during the late stages of infection. Taken together, our results demonstrate that ChSte7 is involved in regulation of vegetative growth, appressorial formation of C. higginsianum, and postinvasive growth in host tissues.

MoARG1, MoARG5,6 and MoARG7 involved in arginine biosynthesis are essential for growth, conidiogenesis, sexual reproduction, and pathogenicity in Magnaporthe oryzae

Arginine is one of the most versatile amino acids in eukaryote cells, which plays important roles in a multitude of processes such as protein synthesis, nitrogen metabolism, nitric oxide (NO) and urea biosynthesis. The de novo arginine biosynthesis pathway is conserved among fungal kingdom, but poorly understood in plant pathogenic fungi. Here, we characterized the functions of three synthetic enzyme-encoding genes MoARG1, MoARG5,6, and MoARG7, which involved the seventh step, second–third step and fifth step of arginine biosynthesis in Magnaporthe oryzae, respectively. Deletion of MoARG1 or MoARG5,6, resulted in arginine auxotrophic mutants, which had a strict requirement for arginine on minimal medium (MM). Both ΔMoarg1 and ΔMoarg5,6 severely reduced in aerial hyphal growth, pigmentation, conidiogenesis, sexual reproduction and pathogenicity. Interestingly, like Saccharomyces cerevisiae, deletion of MoARG7 caused a leaky arginine auxotrophy, and attenuated pathogenicity. Limited appressorium-mediated penetration and restricted invasive hyphae growth in host cells are responsible for the severely attenuated pathogenicity of the Arg− mutants. Additionally, we monitored the NO generation during conidial germination and appressorial formation in both Arg− mutants and wild type, and demonstrated that NO generation may not occur via arginine-dependent pathway in M. oryzae. In summary, MoARG1, MoARG5,6, and MoARG7 are required for growth, conidiogenesis, sexual reproduction, and pathogenicity in M. oryzae.

Endophytic Bacillus subtilis strain E1R-J is a promising biocontrol agent for wheat powdery mildew.

In this study, the biocontrol efficacies of 14 endophytic bacterial strains were tested against Blumeria graminis f. sp. tritici (Bgt) in pot experiments under greenhouse conditions. Bacillus subtilis strain E1R-j significantly reduced disease index and exhibited the best control (90.97%). When different formulations of E1R-j were sprayed 24 h before Bgt inoculation, fermentation liquid without bacterial cell and crude protein suspension displayed the similar effects; and they reduced disease index more than bacterial cell suspension (109 cfu mL−1) and fermentation liquid without protein. The control effects were not significantly different between 1011 and 109 cfu mL−1 of bacterial cell suspension but were higher than 107 cfu mL−1. Further observations showed that conidial germination and appressorial formation of Bgt were retarded by spraying E1R-j 24 h before Bgt inoculation. Compared with the water check, conidial germination and appressorial formation were decreased by 43.3% and 42.7%, respectively. In the treatment with E1R-j, the number of houstoria significantly reduced and the speed of mycelial extension was slowed down in the wheat leaves. Scanning electron microscopy observation revealed that E1R-j significantly suppressed the conidial germination and caused rupture and deformation of germ tubes. On the surface of wheat leaves, mycelia and conidiophores became shrinking.

Prepenetration and Penetration of Colletotrichum gloeosporioides into Guava Fruit (Psidium guajava L.): Effects of Temperature, Wetness Period and Fruit Age

AbstractIn this study, we determined the influences of temperature, wetness period and guava fruit age on infection caused by Colletotrichum gloeosporioides. Optimal temperatures in vitro for germination, appressoria formation and melanization were 22.7, 20.6 and 23°C, respectively. In vivo, the optimal temperatures for germination and appressoria formation were 22.5 and 23°C, respectively. Values for germination, appressoria formation and melanization were higher as the wetness period increased. There was no difference in conidial germination and appressorial formation on fruit of different ages. On the surface of 10‐, 35‐ and 60‐day‐old fruit, despite the high percentage of appressorial formation, there was no development of the penetration peg. Penetration pegs were only observed on the 85‐ and 110‐day‐old fruit. Thickness of the cuticle, size and architecture of epidermal and parenchymal cells, as well as the content of phenolic compounds changed as the fruit ripened.

Fungal growth, proteinaceous toxins and virulence of Pyrenophora teres f. teres on barley

Pyrenophora teres f. teres (Ptt) causes net form net blotch (NFNB), an important disease of barley, but isolates of Ptt vary in their ability to cause symptoms on susceptible cultivars. Ptt isolates with different virulence were used to compare conidial germination and fungal growth on the barley cultivar ‘Sloop’. Whether proteinaceous toxins from culture filtrates of the six isolates or different fractions and sub-fractions of those filtrates induced different symptoms was also investigated. Greater conidial germination and appressorial formation was observed during infection by more virulent isolates but hyphal length was variable. Even though the six isolates varied in virulence from low to high, the proteinaceous toxins extracted from culture filtrates of all isolates were able to induce necrosis when injected into barley leaves. Ptt isolates therefore appear genetically able to produce proteinaceous toxins and the difference in virulence between isolates may reflect the growth of the fungus and the capacity for toxins to be delivered to the plant tissue. Proteins identified in the biologically active fractions included glycoside hydrolase, cysteine hydrolase, CFEM (common in fungal extracellular membrane) domain-containing protein, lactonase and peptidase. These have been previously suggested to have roles in plant cell wall degradation, fungal growth and/or host-pathogen interactions.

Putative RhoGAP proteins orchestrate vegetative growth, conidiogenesis and pathogenicity of the rice blast fungus Magnaporthe oryzae

Rho GTPases, acting as molecular switches, are involved in the regulation of diverse cellular functions. Rho GTPase activating proteins (Rho GAPs) function as negative regulators of Rho GTPases and are required for a variety of signaling processes in cell development. But the mechanisms underlying Rho GAPs in Rho-mediated signaling pathways in fungi are still elusive. There are eight RhoGAP domain-containing genes annotated in the Magnaporthe oryzae genome. To understand the function of these RhoGAP genes, we generated knockout mutants of each of the RhoGAP genes through a homologous recombination-based method. Phenotypic analysis showed that growth rate of aerial hyphae of the Molrg1 deletion mutant decreased dramatically. The ΔMolrg1 mutant showed significantly reduced conidiation and appressorium formation by germ tubes. Moreover, it lost pathogenicity completely. Deletion of another Rho GAP (MoRga1) resulted in high percentage of larger or gherkin-shaped conidia and slight decrease in conidiation. Appressorial formation of the ΔMoRga1 mutant was delayed significantly on hydrophobic surface, while the development of mycelial growth and pathogenicity in plants was not affected. Confocal fluorescence microscopy imaging showed that MoRga1-GFP localizes to septal pore of the conidium, and this localization pattern requires both LIM and RhoGAP domains. Furthermore, either deleting the LIM or RhoGAP domain or introducing an inactivating R1032A mutation in the RhoGAP domain of MoRga1 caused similar defects as the Morga1 deletion mutant in terms of conidial morphology and appressorial formation, suggesting that MoRga1 is a stage-specific regulator of conidial differentiation by regulating some specific Rho GTPases. In this regard, MoRga1 and MoLrg1 physically interacted with both MoRac1-CA and MoCdc42-CA in the yeast two-hybrid and pull-down assays, suggesting that the actions of these two GAPs are involved in MoRac1 and MoCdc42 pathways. On the other hand, six other putative Rho GAPs (MoRga2 to MoRga7) were dispensable for conidiation, vegetative growth, appressorial formation and pathogenicity, suggesting that these Rho GAPs function redundantly during fungal development. Taking together, Rho GAP genes play important roles in M. oryzae development and infectious processes through coordination and modulation of Rho GTPases.

Potential for using crude extract of Sarocladium oryzae for suppression of rice blast

Crude extract of an isolate of the fungus Sarocladium oryzae (CNPAF So 20G), containing the antimicrobial cerulenin, was produced and its antagonistic potential on the rice blast pathogen Magnaporthe oryzae was asessed. Cerulenin was detected in crude extract of S. oryzae through thin layer chromatography showing a Rf value of 0.63. The quantity of cerulenin in the crude extract was 237 µg.mL-1. The in vitro inhibition of germination and appressorial formation of M. oryzae was assessed on an artificial hydrophobic surface, using eight different doses of cerulenin ranging from 0.05 to 30.0 µg.mL-1. The LD50 values calculated based on the Probit-log analysis for germination and appressorial formation were 1.298 ± 0.123 µg.mL-1 and 0.0705 ± 0.0062 µg.mL-1 of cerulenin, respectively. The 30.0 µg.mL-1 of cerulenin dose inhibited 98% and 99%, germination and appressorial formation, respectively. The mode of action of cerulenin was studied by staining conidia with Calcofluor White and fluorescent microscopy showing its effect on plasma membrane. Crude extract of S. oryzae suppressed 63% of rice blast disease in greenhouse conditions. The results indicate that a product based on cerulenin and/or S. oryzae has a great potential to be used in biological control of rice blast.

The Infection Process of Pestalotiopsis Longisetula Leaf Spot on Strawberry Leaves

AbstractPestalotia leaf spot, caused by the fungus Pestalotiopsis longisetula Guba, has become the major disease affecting strawberry production in Brazil. Strawberry seedlings with 4–5 leaves were inoculated with a conidial suspension of P. longisetula (2 × 105 conidia/ml), and leaf samples were collected at 48, 72, 96 and 144 h after inoculation (hai) for observation in the scanning electron microscope. Conidia germinated within 48 hai. At 72 hai, conidia had formed very long germ tubes over the epidermal cells without any evidence of appressorial formation nor direct penetration. At 96 hai, fungal hyphae grew inter‐ and intracellularly in the lacunous parenchyma and also through tracheary elements. Pycnidia were first observed on the leaf surface at 96 hai. At 144 hai, conidia of P. longisetula were first liberated from the pycnidia. This study adds new information to better understand of the infection process of P. longisetula that may help in developing more effective disease control strategies.

Increased enzymatic activity in rice leaf blast suppression by crude extract of Epicoccum sp

Epicoccum sp. showed in vitro antagonism to the rice pathogens Magnaporthe oryzae, Rhizoctonia solani, Sarocladium oryzae, Monographella albescens and Cochliobolus miyabeanus in dual culture. The colony growth of the rice blast fungus, M. oryzae, was reduced by 42.50%. The lethal doses (LD50) determined based on probit-log dosage response curves at 3 and 6 hours after germination were 393.0 and 326.6 ppm, respectively. The crude extract (600 ppm) reduced appressorial formation by 95.68%. A greenhouse experiment comparing the relative efficiency of conidial suspension and crude extract of Epicoccum sp. in the suppression of leaf blast showed no statistical difference between both application methods. However, the crude extract of Epicoccum sp. (4000 ppm) 48 hours before the application of M. oryzae induced resistance and suppressed leaf blast by 97.6%. Scanning electron microscopy of rice leaves inoculated with crude extract of Epicoccum sp. and challenged with M. oryzae showed appressorial deformation on penetration phase. Peroxidase and β-1,3-glucanase activities increased in plants sprayed with crude extract of Epicoccum sp., 24 hours after the application of the challenger. PAL as well as chitinase activities increased 72 hours after challenge inoculation. Epicoccum sp. was shown to be a potential antagonist and inducer of resistance against M. oryzae.

Adjuvant and Refined Corn Oil Formulation Effects on Conidial Germination, Appressorial Formation and Virulence of the Bioherbicide, Colletotrichum truncatum

Several surfactants, plant extracts, and fatty acids were tested for stimulation of conidial germination and appressorial formation of Colletotrichum truncatum, a bioherbicide of the weed, hemp sesbania (Sesbania exaltata). The commercial surfactants (Tweens® 40, 60, 80, 85 and Myvatex® 60) at concentrations of 0.25 to 1.0 % (v/v), stimulated germination, but these effects were not exclusively related to their hydrophylic-lipophylic balance values. The stimulatory surfactants were also tested for germination and virulence of C. truncatum when formulated with the conidia and applied to hemp sesbania seedlings. Conidia formulated in either water, a surfactant or, an emulsion of refined corn oil were ineffective on the plant in the absence of dew or when dew was delayed. However, formulations of conidia combined with surfactant in emulsified refined corn oil did exhibit bioherbicidal activity when adequate dew or free-moisture was unavailable. This is important since previous reports indicated that refined corn oil did not enhance bioherbicidal activity, whereas unrefined corn oil promoted pathogen germination and efficacy of C. truncatum. The fatty acids tested had little or no effect on conidial germination but, aqueous extracts of several plant species including pathogen hosts and non-hosts, stimulated germination. Appressorial formation influenced by the surfactants did not necessarily reflect the disease rating on hemp sesbania seedlings. Overall, results show that formulations containing an emulsion of conidia, a surfactant and refined corn oil have potential for enhancement of the efficacy of C. truncatum.

Infection of Brachypodium distachyon by formae speciales of Puccinia graminis: early infection events and host-pathogen incompatibility.

Puccinia graminis causes stem rust, a serious disease of cereals and forage grasses. Important formae speciales of P. graminis and their typical hosts are P. graminis f. sp. tritici (Pg-tr) in wheat and barley, P. graminis f. sp. lolii (Pg-lo) in perennial ryegrass and tall fescue, and P. graminis f. sp. phlei-pratensis (Pg-pp) in timothy grass. Brachypodium distachyon is an emerging genetic model to study fungal disease resistance in cereals and temperate grasses. We characterized the P. graminis-Brachypodium pathosystem to evaluate its potential for investigating incompatibility and non-host resistance to P. graminis. Inoculation of eight Brachypodium inbred lines with Pg-tr, Pg-lo or Pg-pp resulted in sporulating lesions later accompanied by necrosis. Histological analysis of early infection events in one Brachypodium inbred line (Bd1-1) indicated that Pg-lo and Pg-pp were markedly more efficient than Pg-tr at establishing a biotrophic interaction. Formation of appressoria was completed (60–70% of germinated spores) by 12 h post-inoculation (hpi) under dark and wet conditions, and after 4 h of subsequent light exposure fungal penetration structures (penetration peg, substomatal vesicle and primary infection hyphae) had developed. Brachypodium Bd1-1 exhibited pre-haustorial resistance to Pg-tr, i.e. infection usually stopped at appressorial formation. By 68 hpi, only 0.3% and 0.7% of the Pg-tr urediniospores developed haustoria and colonies, respectively. In contrast, development of advanced infection structures by Pg-lo and Pg-pp was significantly more common; however, Brachypodium displayed post-haustorial resistance to these isolates. By 68 hpi the percentage of urediniospores that only develop a haustorium mother cell or haustorium in Pg-lo and Pg-pp reached 8% and 5%, respectively. The formation of colonies reached 14% and 13%, respectively. We conclude that Brachypodium is an apt grass model to study the molecular and genetic components of incompatiblity and non-host resistance to P. graminis.

Ultrastructural Changes of Sphaerotheca fuliginea (Schlechtend.:Fr.) Pollacci in Cucumber After Treated by Chrysophanol

Chrysophanol (1,8-dihydroxy-3-methylanthraquinone) is a free anthraquinone compound and a secondary metabolite of medicinal plant rhubarb. Chrysophanol has been reported to have both protective and curative activity against Sphaerotheca fuliginea (Schlechtend.:Fr.) Pollacci, the causal agent of cucumber powdery mildew. In this paper the ultrastructure of powdery mildew on cucumber leaves was studied using electron microscopy after the leaves were treated with chrysophanol. Results showed that preventive treatments with chrysophanol affected fungal development, including spore germination, appressorial formation, and penetration. In the curative treatment, chrysophanol affected fungal survival, resulting in broken cell wall of germ tubes, swelling and collapse of hyphal tips, hyphal malformation, delayed and reduced sporulation. The morphological changes induced by chrysophanol at the ultrastructural level were reflected by haustorium deformation, vacuolization, abortion, and necrosis. Host cell walls infected or adjacent to haustoria were thickened. All these morphological changes of S. fuliginea further confirmed the fungicidal activity of chrysophanol on powdery mildew of cucumber.

Race-specific and ulvan-induced defense responses in bean (Phaseolus vulgaris) against Colletotrichum lindemuthianum

The present work aimed to monitor and compare race-specific and ulvan-induced defense responses to race 73 of Colletotrichum lindemuthianum (Cl) in resistant and susceptible bean plants. Under greenhouse conditions, foliar spraying of ulvan, a water-soluble algal polysaccharide, reduced anthracnose severity locally and systemically in susceptible plants by 60 and 40% respectively. Neither race-specific resistance nor ulvan treatment affected both conidial germination and appressorial formation of Cl on leaves. Race 73-specific resistance was associated with a rapid recognition of the pathogen, expressed by a more frequent hypersensitive response in bean epidermal cells. Ulvan increased peroxidase activity in resistant, but not in susceptible plants, and glucanase activity in both resistant and susceptible plants inoculated or not with Cl. This is the first report of an increase in activity of plant defense-related enzymes by treatment of a polysaccharide before pathogen infection.