Actin expression in germinating seeds of Phaseolus vulgaris L.

Actin was present at very low levels in the seeds of common bean (Phaseolus vulgaris L.) compared with those from other species, and was observed mostly in the embryo. A time-course of actin expression in germinating bean seeds revealed an induced expression of both the mRNA and protein. Initially, the actin mRNA in seeds was barely detectable by northern blot analysis. However, there was a substantial increase in the expression of the actin mRNA at 24, 48 and 72 h after imbibition, compared with an internal control consisting of a late-embryogenesis-abundant (LEA) type IV gene from P. vulgaris. An increase in the amount of actin in total seed extracts that parallelled that of the mRNA was detected by western blotting starting at 24 h after imbibition. This increase was more apparent when the embryo alone was analyzed. Two-dimensional western blots initially revealed three actin isoforms with isoelectric points (pIs) of approximately 5.6, 5.7 and 5.8, the amounts of which increased within a 48-h period, when a new minor isoform of pI approximately 5.5 appeared; however, after 72 h, the pI-5.8 isoform had almost disappeared and the pI-5.5 isoform had disappeared completely, indicating that these two minor isoforms are expressed transiently. These results indicate that actin is at very low levels in the dry seed but undergoes an increased and differential expression during imbibition, an event probably required to carry out all the necessary functions for germination. Received: 21 July 1998 / Accepted: 1 September 1998     

In Planta Production of Indole-3-Acetic Acid by Colletotrichum gloeosporioides f. sp. aeschynomene

The plant pathogenic fungus Colletotrichum gloeosporioides f. sp. aeschynomene utilizes external tryptophan to produce indole-3-acetic acid (IAA) through the intermediate indole-3-acetamide (IAM). We studied the effects of tryptophan, IAA, and IAM on IAA biosynthesis in fungal axenic cultures and on in planta IAA production by the fungus. IAA biosynthesis was strictly dependent on external tryptophan and was enhanced by tryptophan and IAM. The fungus produced IAM and IAA in planta during the biotrophic and necrotrophic phases of infection. The amounts of IAA produced per fungal biomass were highest during the biotrophic phase. IAA production by this plant pathogen might be important during early stages of plant colonization.     

Expression of a glycogen synthase protein kinase homolog from Colletotrichum gloeosporioides f.sp. malvae during infection of Malva pusilla

The potential role of a GSK3 protein kinase homolog, cggsk, was examined from Colletotrichum gloeosporioides f.sp. malvae, a fungal pathogen of Malva pusilla. A peak in cggsk expression relative to a constitutively expressed fungal actin gene occurred during host penetration and was followed by much lower expression levels during subsequent biotrophic and necrotrophic growth in host tissue. The peak level of cggsk expression observed during penetration was 21-fold greater than that during necrotrophic growth. Expression of cggsk showed small but reproducible changes during growth in culture; however, the levels were always similar to that during necrotrophic growth in the host. One possible role for cggsk could be to coordinate fungal development during host penetration.     

A rice gene encoding glycosyl hydrolase plays contrasting roles in immunity depending on the type of pathogens

Because pathogens use diverse infection strategies, plants cannot use one-size-fits-all defence and modulate defence responses based on the nature of pathogens and pathogenicity mechanism. Here, we report that a rice glycoside hydrolase (GH) plays contrasting roles in defence depending on whether a pathogen is hemibiotrophic or necrotrophic. The Arabidopsis thaliana MORE1 (M agnaporthe o ryzae re sistance 1) gene, encoding a member of the GH10 family, is needed for resistance against M. oryzae and Alternaria brassicicola, a fungal pathogen infecting A. thaliana as a necrotroph. Among 13 rice genes homologous to MORE1, 11 genes were induced during the biotrophic or necrotrophic stage of infection by M. oryzae. CRISPR/Cas9-assisted disruption of one of them (OsMORE1a) enhanced resistance against hemibiotrophic pathogens M. oryzae and Xanthomonas oryzae pv. oryzae but increased susceptibility to Cochliobolus miyabeanus, a necrotrophic fungus, suggesting that OsMORE1a acts as a double-edged sword depending on the mode of infection (hemibiotrophic vs. necrotrophic). We characterized molecular and cellular changes caused by the loss of MORE1 and OsMORE1a to understand how these genes participate in modulating defence responses. Although the underlying mechanism of action remains unknown, both genes appear to affect the expression of many defence-related genes. Expression patterns of the GH10 family genes in A. thaliana and rice suggest that other members also participate in pathogen defence.     

Tryptophan‐derived secondary metabolites in Arabidopsis thaliana confer non‐host resistance to necrotrophic Plectosphaerella cucumerina fungi

A defence pathway contributing to non‐host resistance to biotrophic fungi in Arabidopsis involves the synthesis and targeted delivery of the tryptophan (trp)‐derived metabolites indol glucosinolates (IGs) and camalexin at pathogen contact sites. We have examined whether these metabolites are also rate‐limiting for colonization by necrotrophic fungi. Inoculation of Arabidopsis with adapted or non‐adapted isolates of the ascomycete Plectosphaerella cucumerina triggers the accumulation of trp‐derived metabolites. We found that their depletion in cyp79B2 cyp79B3 mutants renders Arabidopsis fully susceptible to each of three tested non‐adapted P. cucumerina isolates, and super‐susceptible to an adapted P. cucumerina isolate. This assigns a key role to trp‐derived secondary metabolites in limiting the growth of both non‐adapted and adapted necrotrophic fungi. However, 4‐methoxy‐indol‐3‐ylmethylglucosinolate, which is generated by the P450 monooxygenase CYP81F2, and hydrolyzed by PEN2 myrosinase, together with the antimicrobial camalexin play a minor role in restricting the growth of the non‐adapted necrotrophs. This contrasts with a major role of these two trp‐derived phytochemicals in limiting invasive growth of non‐adapted biotrophic powdery mildew fungi, thereby implying the existence of other unknown trp‐derived metabolites in resistance responses to non‐adapted necrotrophic P. cucumerina. Impaired defence to non‐adapted P. cucumerina, but not to the non‐adapted biotrophic fungus Erysiphe pisi, on cyp79B2 cyp79B3 plants is largely restored in the irx1 background, which shows a constitutive accumulation of antimicrobial peptides. Our findings imply differential contributions of antimicrobials in non‐host resistance to necrotrophic and biotrophic pathogens.     

The biotrophy-associated secreted protein 4 (BAS4) participates in the transition of Magnaporthe oryzae from the biotrophic to the necrotrophic phase

The physiological and metabolic processes of host plants are manipulated and remodeled by phytopathogenic fungi during infection, revealed obvious signs of biotrophy of the hemibiotrophic pathogen. As we known that effector proteins play key roles in interaction of hemibiotrophic fungi and their host plants. BAS4 (biotrophy-associated secreted protein 4) is an EIHM (extrainvasive hyphal membrane) matrix protein that was highly expressed in infectious hyphae. In order to study whether BAS4 is involved in the transition of rice blast fungus from biotrophic to necrotrophic phase, The susceptible rice cultivar Lijiangxintuanheigu (LTH) that were pre-treated with prokaryotic expression product of BAS4 and then followed with inoculation of the blast strain, more serious blast disease symptom, more biomass such as sporulation and fungal relative growth, and lower expression level of pathogenicity-related genes appeared in lesion of the rice leaves than those of the PBS-pretreated-leaves followed with inoculation of the same blast strain, which demonstrating that BAS4 invitro changed rice defense system to facilitate infection of rice blast strain. And the susceptible rice cultivar (LTH) were inoculated withBAS4-overexpressed blast strain, we also found more serious blast disease symptom and more biomass also appeared in lesion of leaves inoculated with BAS4-overexpressed strain than those of leaves inoculated with the wild-type strain, and expression level of pathogenicity-related genes appeared lower in biotrophic phase and higher in necrotrophic phase of infection, indicating BAS4 maybe in vivo regulate defense system of rice to facilitate transition of biotrophic to necrotrophic phase. Our data demonstrates that BAS4 in vitro and in vivo participates in transition from the biotrophic to the necrotrophic phase of Magnaporthe oryzae.     

Finalizing host range determination of a weed biological control pathogen with best linear unbiased predictors and damage assessment

Colletotrichum gloeosporioides f. sp. salsolae (Penz.) Penz. & Sacc. in Penz. (CGS) is a facultative parasitic fungus being evaluated as a classical biological control agent of Russian thistle or tumbleweed (Salsola tragus L.). In initial host range determination tests, Henderson’s mixed model equations (MME) were used to generate best linear unbiased predictors (BLUPs) of disease severity reaction to CGS among 89 species of plants related to S. tragus. The MME provided: (1) disease assessments for rare and difficult or impossible to grow species, (2) environmentally independent measures of disease severity, (3) measures of disease severity for species versus a sample of material tested in a greenhouse, (4) objective indicators of susceptible and non-susceptible species, (5) a means to objectively compare disease on targets versus non-targets. Of the 89 species evaluated by the MME, eight native N. American species were predicted to be susceptible. As a result of these predictions, these eight species were further evaluated to determine the amount of actual damage caused by CGS. This was done by comparing root and shoot areas and weights between non-inoculated plants and plants inoculated with CGS. Results showed that several of the species exhibited some minor reduction in root weight and root area, but none of the species had any damage to above-ground plant parts. This supports the BLUP output in the initial host range determination tests. As a result of both analyses, there is no evidence that CGS would cause any non-target effects in nature.     

A conserved GH17 glycosyl hydrolase from plant pathogenic Dothideomycetes releases a DAMP causing cell death in tomato

To facilitate infection, pathogens deploy a plethora of effectors to suppress basal host immunity induced by exogenous microbe-associated or endogenous damage-associated molecular patterns (DAMPs). In this study, we have characterized family 17 glycosyl hydrolases of the tomato pathogen Cladosporium fulvum (CfGH17) and studied their role in infection. Heterologous expression of CfGH17-1 to 5 by potato virus X in different tomato cultivars showed that CfGH17-1 and CfGH17-5 enzymes induce cell death in Cf-0, Cf-1 and Cf-5 but not in Cf-Ecp3 tomato cultivars or tobacco. Moreover, CfGH17-1 orthologues from other phytopathogens, including Dothistroma septosporum and Mycosphaerella fijiensis, also trigger cell death in tomato. CfGH17-1 and CfGH17-5 are predicted to be β-1,3-glucanases and their enzymatic activity is required for the induction of cell death. CfGH17-1 hydrolyses laminarin, a linear 1,3-β-glucan with 1,6-β linkages. CfGH17-1 expression is down-regulated during the biotrophic phase of infection and up-regulated during the necrotrophic phase. Deletion of CfGH17-1 in C. fulvum did not reduce virulence on tomato, while constitutive expression of CfGH17-1 decreased virulence, suggesting that abundant presence of CfGH17-1 during biotrophic growth may release a DAMP that activates plant defence responses. Under natural conditions CfGH17-1 is suggested to play a role during saprophytic growth when the fungus thrives on dead host tissue, which is in line with its high levels of expression at late stages of infection when host tissues have become necrotic. We suggest that CfGH17-1 releases a DAMP from the host cell wall that is recognized by a yet unknown host plant receptor.     

Identification and characterization of Cercospora beticola necrosis-inducing effector CbNip1

Cercospora beticola is a hemibiotrophic fungus that causes cercospora leaf spot disease of sugar beet (Beta vulgaris). After an initial symptomless biotrophic phase of colonization, necrotic lesions appear on host leaves as the fungus switches to a necrotrophic lifestyle. The phytotoxic secondary metabolite cercosporin has been shown to facilitate fungal virulence for several Cercospora spp. However, because cercosporin production and subsequent cercosporin-initiated formation of reactive oxygen species is light-dependent, cell death evocation by this toxin is only fully ensured during a period of light. Here, we report the discovery of the effector protein CbNip1 secreted by C. beticola that causes enhanced necrosis in the absence of light and, therefore, may complement light-dependent necrosis formation by cercosporin. Infiltration of CbNip1 protein into sugar beet leaves revealed that darkness is essential for full CbNip1-triggered necrosis, as light exposure delayed CbNip1-triggered host cell death. Gene expression analysis during host infection shows that CbNip1 expression is correlated with symptom development in planta. Targeted gene replacement of CbNip1 leads to a significant reduction in virulence, indicating the importance of CbNip1 during colonization. Analysis of 89 C. beticola genomes revealed that CbNip1 resides in a region that recently underwent a selective sweep, suggesting selection pressure exists to maintain a beneficial variant of the gene. Taken together, CbNip1 is a crucial effector during the C. beticola–sugar beet disease process.     

Larval cannibalism in aphidophagous ladybirds: Influencing factors, benefits and costs

Spray retention on scentless chamomile and round-leaved mallow was characterized in relation to droplet size and travel speed. The effect of varying retention on biocontrol efficacy was studied using Colletotrichum truncatum and Colletotrichum gloeosporioides f. sp. malvae, the bioherbicide agents for the two weeds, respectively. In retention studies, a tracer dye solution was applied using a cabinet sprayer fitted with a fine, medium, or coarse nozzle at travel speeds of 0.5, 1.0, and 2.0 km/h that resulted in approximate application volumes of 500–2000 L/ha. Retention efficiency, a ratio between the volume retained on plants and actual volume applied, was calculated for each application. In general, finer sprays achieved higher retention efficiency on whole plants of both weeds. On an average, fine sprays resulted in 68% greater retention than coarse sprays on scentless chamomile and 22–59% on round-leaved mallow. Faster travel speeds increased spray retention only slightly. Applying the biocontrol agents at 500 L/ha with different droplet sizes showed different effects on weed control on scentless chamomile and round-leaved mallow. C. truncatum applied with fine droplets was more efficacious than treatments with coarser droplets on scentless chamomile. On round-leaved mallow, however, the efficacy of C. gloeosporioides f. sp. malvae did not appear to be substantially affected by the droplet size. This varying effect may be due to reduced retention on the vertical stems of round-leaved mallow, which is critical to biocontrol of this weed by C. gloeosporioides f.sp. malvae.     

Bestimmung pektolytischer Enzyme bei mehreren Schadpilzen von Citrusfrüchten

Characterization of pectolytic enzymes From several fungi causing post-harvest decay of citrus fruits From culture filtrates of Penicillium digitatum Sacc., Trichoderma viride Penz., Phomopsis citri (Faw.) cf. Diaporthe citri (Faw.) Wolf, and Colletotrichum gloeosporioides Penz. endo-polygalacturonase and endo-polymethylgalacturonase have been isolated. Both enzymes were active under acid conditions only. The last named fungus additionally produced pectic acid-transeliminase, the activity was found under alkaline conditions.     

The hemibiotrophic lifestyle of Colletotrichum species

Colletotrichum species infect several economically important crop plants. To establish a compatible parasitic interaction, a specialized infection cell, the melanized appressorium, is differentiated on the cuticle of the host. After penetration, an infection vesicle and primary hyphae are formed. These structures do not kill the host cell and show some similarities with haustoria formed by powdery mildews and rust fungi. Therefore, this stage of infection is called biotrophic. Later in the infection process, necrotrophic secondary hyphae spread within and kill the host tissue. The lifestyle of Colletotrichum species is called hemibiotrophic, as biotrophic and necrotrophic developmental stages are sequentially established. As most Colletotrichum species are accessible to molecular techniques, genes can be identified and functionally characterized. Here we demonstrate that Agrobacterium tumefaciens-mediated transformation is a well-suited method for tagging of genes mediating compatibility in the Colletotrichum graminicola–maize interaction.     

Genetic and physical analysis of a YAC contig spanning the fungal disease resistance locus Asc of tomato (Lycopersicon esculentum)

The Alternaria stem canker disease of tomato is caused by the necrotrophic fungal pathogen Alternaria alternata f. sp. lycopersici (AAL). The fungus produces AAL toxins that kill the plant tissue. Resistance to the fungus segregates as a single locus, called Asc, and has been genetically mapped on chromosome 3 of tomato. We describe here the establishment of a 1383-kb YAC contig covering the Asc locus and a series of plants selected for recombination events around the Asc locus. It was shown that the YAC contig corresponds to a genetic distance of at least 11.2 cM. Thus, the recombination rate in the Asc region is six times higher (123 kb/cM) than the average for the tomato genome. Furthermore, the Asc locus could be localised to a 91-kb fragment, thus paving the way for the cloning and identification of the Asc gene(s) by complementation. Received: 31 July 1998 / Accepted: 6 October 1998