An Aqueous Extract of the Dry Mycelium of Penicillium chrysogenum Induces Resistance in Several Crops under Controlled and Field Conditions

We have examined the effect of Pen, an aqueous extract of the dry mycelium of Penicillium chrysogenum, on plant–pathogen interactions. Pen controlled a broad range of pathogens on several crop plants under greenhouse and field conditions. Pen protected grapevine from downy and powdery mildew (caused by Plasmopara viticola and Uncinula necator), tomato from early blight (caused by Phytophthora infestans), onion from downy mildew (Peronospora destructor) and apple trees from apple scab (caused by Venturia inaequalis) to a similar extent as fungicides such as copper and sulphur or well-known inducers such as benzothiadiazole or β-aminobutyric acid. Pen had no major direct fungicidal effect and is thus supposed to protect plants by activating their defense mechanisms. The raw material for extraction of Pen was available in constant quality, a prerequisite for commercial application. Under certain conditions, Pen caused phytotoxic side effects. The symptoms mostly consisted of small necrotic spots or, more rarely, of larger necrotic areas. The development of the symptoms was dependent on several parameters, including concentration of Pen, the number of applications, the persistence on the plant tissue, the plant species and variety and environmental conditions. In grapevine, a partially purified fraction of Pen was much less toxic than the crude Pen extract, but protected the plants to a similar extent against P. viticola. Our data show that Pen has interesting and unique properties as a plant protection agent, but more research is needed to further reduce its phytotoxic side effects.     

Characterization of an antibacterial substance produced by <Emphasis Type="Italic">Erwinia carotovora</Emphasis> subsp. <Emphasis Type="Italic">carotovora</Emphasis> Ecc 32

A total of 88 strains of Erwinia carotovora subsp. carotovora (Ecc) isolated from various host plants in several geographic regions were screened for production of antibacterial substances using the same strains as indicators. Of the 88 strains, 72 produced antibacterial substances. One of these 72 strains, a Brazilian strain Ecc 32, produced an antibacterial substance active against all tested Ecc strains on TSA medium. The antibacterial spectrum of the compound from Ecc 32 strain was limited to closely related strains of soft-rot Erwinia species. Such a narrow spectrum of activity is typical of bacteriocins. The compound produced by Ecc 32 strain, however, was resistant to some enzymes and detergents. Moreover, the compound was heat-stable and active over a wide pH range. The physical characteristics of the compound were not in agreement with those of bacteriocin or carotovoricin.     

Characterization of Pectolytic Erwinias as Highly Sophisticated Pathogens of plants

Erwinia carotovora and Erwinia chrysanthemi are the two most important soft rotting bacteria of commercially-grown plants. They are genetically diverse as is evident from polymorphisms in the pel and recA genes as well as in rrn, the ribsomal gene cluster. Subpopulations grouped into biovars, pathovars, or subspecies associated with various hosts and in different geographic regions suggest specialization in host preference and/or survival in diverse environments. Previous characterization of the pectolytic erwinias as opportunistic pathogens is being replaced by a realization that this group of bacteria exhibits a sophisticated repertoire of pathogenicity and virulence genes and regulators. The presence of an entire hrp gene cluster and associated type III secretion system, and global regulators which regulate virulence determinants such as exoenzyme production and motility, attest to a highly specialized pathogen. The fact that production of extracellular plant cell wall-degrading enzymes are coordinately activated by the diffusible signal molecule N-acyl-homoserine lactone in a population density-dependent manner may explain the occurrence of pectolytic erwinia in asymptomatic plant tissues. Transgenic plants expressing bacterial quorum-sensing signal molecules modulate this sensory system and exhibit resistance to soft rot infection. The pectolytic erwinias, being significant plant pathogens that are neither of quarantine concern nor a human health hazard while readily isolated from field sources, make an ideal model for investigating the genetic basis of plant pathogenesis and environmental fitness.     

A Survey of Trunk Disease Pathogens within Rootstocks of Grapevines in Spain

Grapevine trunk disease pathogens, and specifically Petri disease pathogens, can be spread by planting infected plants. Due to the increasing incidence of Petri disease and other young grapevine declines reported lately in Spain, a sampling of plants used before for new vineyards were carried out in 2002 and 2004. A total number of 208 plants (grafted and non grafted) were collected, of which 94 plants (45.2%) were infected with at least one of the following pathogens: Phaeomoniella chlamydospora, and species of Phaeoacremonium, Botryosphaeria, Cylindrocarpon, and Phomopsis. Species of the genera Phaeoacremonium and Botryosphaeria isolated in 2004 were identified using morphological and molecular characters. Species of Phaeoacremonium identified were P. aleophilum and P. parasiticum; and those of Botryosphaeria were B. obtusa, B. dothidea and B. parva. This is the first report of P. parasiticum and B. parva occurring on grapevines in Spain. Distribution of pathogens within the plants was studied in 2004. Phaeomoniella chlamydospora was not detected in the graft union of any plant; however, species of Botryosphaeria and Phomopsis were detected along the plant, but mainly in the graft union; Phaeoacremonium aleophilum was detected along the grafted plants, but not in rooted rootstocks. The results suggest that infected plants used for new plantings in Spain are an important source of primary inoculum of the pathogens associated with grapevine trunk diseases in the field.     

A PAL1 Gene Promoter–Green Fluorescent Protein Reporter System to Analyse Defence Responses in Live Cells of Arabidopsis thaliana

Arabidopsis thaliana ecotype Columbia-0 was transformed with a green fluorescent protein (GFP) gene under control of a phenylalanine ammonia-lyase (PAL) promoter. PAL is a key enzyme of the phenylpropanoid pathway and is induced to high levels during plant stress. Constitutive expression of PAL1 promoter-controlled GFP occurred in vascular tissues within stems, leaves and roots and in developing flowers. PAL1 promoter–GFP expression was examined in leaves of transgenic plants subjected to an abiotic elicitor, mechanical wounding or to inoculation with the pathogens Pseudomonas syringae pv. tomato or Peronospora parasitica. Wounding of leaves and treatment with an abiotic elicitor and compatible interactions produced low to moderate levels of GFP. However, in incompatible interactions there were high levels of GFP produced. In incompatible interactions, the intensity of GFP fluorescence was similar to that produced in transgenic plants expressing GFP driven by the CaMV promoter. The bright green fluorescence produced in live cells and tissues was readily visualised using conventional fluorescence microscopy and was quantified using spectroflourometry. This is the first report of the use of GFP as a reporter of defence gene activation against pathogens. It has several advantages over other reporter genes including real time analysis of gene expression and visualisation of defence gene activation in a non-invasive manner.     

N-terminal domain including conserved flg22 is required for flagellin-induced hypersensitive cell death in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Flagellin in Pseudomonas syringae is a potent elicitor of defense responses including hypersensitive cell death in dicot plants. The oligopeptides flg22 consisting of 22 conserved amino acids near the N-terminus of flagellins is reported to induce plant defense responses. Because glycosylation of the central domain of flagellin affects its elicitor activity, we investigated whether any peptide sequence in addition to flg22 is required for flagellin-induced hypersensitive reaction. A study of recombinant flagellin polypeptides indicated that the N-terminal domain including the conserved flg22 is required for flagellin-induced hypersensitive cell death in Arabidopsis thaliana.     

Flagella-mediated motility is required for biofilm formation by Erwinia carotovora subsp. carotovora

To elucidate the role of flagella in biofilm formation by Erwinia carotovora subsp. carotovora EC1, we used a nonflagellate, nonmotile mutant (ΔfliC) and a flagellate, nonmotile mutant (ΔmotA). A biofilm-inducing medium, which contains the yeast peptone (YP) medium plus the salts of M-63 minimal medium, supported biofilm formation to a greater extent than either the YP or Luria Bertani (LB) medium alone. We demonstrated that both the ΔfliC and ΔmotA mutants greatly reduced their ability to form a biofilm on the surface of the wells of polyvinyl chloride (PVC) microtiter plates. The inability of both mutants to form biofilm on the PVC surface was further confirmed with phase-contrast microscopy. Both aflagellate (ΔfliC) and flagellate (ΔmotA) nonmotile mutants were equally defective in attachment to the PVC surface. The treatment of bacteria with the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP), which inhibits the motility of this organism, reduced greatly the biofilm formation. Based on these results, flagella-mediated motility may play an important role in biofilm formation of E. carotovora subsp. carotovora EC1.     

Characterization and differentiation of <Emphasis Type="Italic">Erwinia carotovora</Emphasis> strains from mulberry trees

We recently reported that two diverse types (types 1 and 2) were identified among strains of Erwinia carotovora from mulberry trees. Type 1 strains were similar to E. carotovora subsp. carotovora (Ecc), whereas type 2 strains were distinct from Ecc and other E. carotovora strains. In this study, seven more mulberry strains of type 2 and reference strains were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and randomly amplified of polymorphic DNA (RAPD). On the basis of SDS-PAGE profiles of whole-cell proteins, type 2 strains had high similarity with one another. In addition, they had an unique peptide band with a molecular mass of approximately 28kDa. RAPD analysis showed that they were also effectively differentiated by a strong, specific RAPD fragment for type 2 strains. Based on these two approaches, we have confirmed that the present type 2 strains from mulberry can be discriminated clearly from other soft rot Erwinia species.     

An oligonucleotide probe for the detection of Erwinia herbicola and Erwinia ananas

An oligonucleotide probe targeting the rRNA of Erwinia herbicola and Erwinia ananas was designed to detect their cells using fluorescence in situ hybridization. The Cy3-labeled probe hybridized strongly with these species but very weakly with nontarget species such as Erwinia mallotivora, Erwinia nigrifluens, Erwinia cypripedii, Erwinia chrysanthemi, Erwinia carotovora subsp. carotovora, E. carotovora subsp. atroseptica, and Escherichia coli. This technique visualized E. herbicola cells after inoculation of kumquat fruits. The probe is promising as a tool for studying population dynamics of E. herbicola and E. ananas.     

Aetiology and causal agents of mango sudden decline disease in the Sultanate of Oman

Mango sudden decline is a recently introduced, economically serious disease in Oman. Affected mango trees have wilting symptoms that usually begin on one side and later spread to involve the entire tree. Trees exude amber-coloured gum from the bark of their trunks or branches and vascular tissues are discoloured. Having entered Oman in the recent past, survey data is presented that shows the disease to have spread throughout the northern part of the country. Evidence is presented that the vascular wilt pathogen Ceratocystis fimbriata causes mango sudden decline disease in Oman, possibly in concert with Lasiodiplodia theobromae and the recently described Ceratocystis omanensis. Isolates of these fungi from affected trees, cause infection and can be recovered from inoculated seedlings. Bark beetles (Hypocryphalus mangiferae) are shown to carry C. fimbriata and L. theobromae and are presumably responsible for transmitting both pathogens to healthy mango trees. Acting as a wounding agent and vector, the bark beetle is likely to have assisted the rapid spread of the disease across Oman.     

Mycorrhiza induced resistance in potato plantlets challenged by Phytophthora infestans

Biological control of soil-borne pathogens by arbuscular mycorrhizal (AM) fungi has been repeatedly demonstrated. However, their role in the control of above-ground hemibiotrophic pathogens is less conclusive. Here, we investigated in vitro the impact of an AM fungus on Phytophthora infestans in potato plants. The leaf infection index was decreased in mycorrhizal potato plants. Real-Time Quantitative PCR revealed the induction of two pathogenesis related genes (PR1 and PR2) in the leaves of mycorrhizal plants shortly after infection with P. infestans. These results suggested a systemic resistance in mycorrhizal plants, related to the priming of the two PR genes in potato.     

Involvement of NbNOA1 in NO production and defense responses in INF1-treated Nicotiana benthamiana

During defense responses, plant cells produce nitric oxide (NO), which may control many physiological processes. In a previous study, we reported that nitrate reductase (NR) is responsible in part for INF1 elicitor-induced NO production in Nicotiana benthamiana, but the possibility remains that other NO-generating system(s) contribute to NO production. In mammalian cells, NO production is catalyzed by NO synthase (NOS). However, NOS-like enzyme(s) have never been identified in plants, and only the gene for Arabidopsis thaliana nitric oxide-associated 1 (AtNOA1) has been identified as a putative regulator of NOS activity in plants. In this study, we cloned NbNOA1, a homolog of AtNOA1, from N. benthamiana and investigated its involvement in NO production induced by INF1. The NbNOA1 gene was silenced by a virus-induced gene-silencing (VIGS) technique. NbNOA1-silenced plants had yellowish leaves. Silencing NbNOA1 partially decreased INF1-induced NO production, while overexpressing NbNOA1 did not affect NO production. Silencing NbNOA1 suppressed INF1-induced PR1a gene expression and increased susceptibility to Colletotrichum lagenarium. These results suggest that NbNOA1 is involved in INF1-mediated NO production and is required for defense responses. The nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under accession number AB303300.     

Induction of a defense response in strawberry mediated by an avirulent strain of <Emphasis Type="Italic">Colletotrichum</Emphasis>

In the strawberry crop area of Tucumán (north-west Argentina) the three species of Colletotrichum causing anthracnose disease (C. acutatum, C. fragariae and C. gloeosporioides) were detected. Among all isolates characterized, one of them identified as C. acutatum (M11) and another as C. fragariae (F7) were selected due to their conspicuous interaction with the strawberry cultivar Pájaro. Whereas isolate M11 produced a strong compatible interaction in cv. Pájaro with clear disease symptoms (DSR = 5.0), the isolate F7 brought about a typical incompatible interaction (DSR = 1.0). When plants of cv. Pájaro were inoculated with F7 prior to the inoculation with M11, the former avirulent strain prevented the growth of the latter virulent pathogen. Experimental evidence indicated that the time elapsed between the first inoculation with the avirulent pathogen and the second inoculation with the virulent one was crucial to inhibit the growth of the latter. The growth of F7 on the plant without provoking damage and the fact that there was no in vitro antagonistic effect between the pathogens, suggests that the avirulent strain triggers a plant defensive response against M11. The defense response was further confirmed by the detection of an early oxidative burst occurring within 4 h after the first inoculation and by the observation of anatomical changes associated with defense mechanisms that lasted 50 days after the inoculation with F7. Results obtained support the hypothesis that the plant resistance against the virulent strain M11 is elicited by one or more diffusible(s) compound(s) produced by the avirulent strain F7.     

Induction of peroxidase, scopoletin, phenolic compounds and resistance in Hevea brasiliensis by elicitin and a novel protein elicitor purified from Phytophthora palmivora

Elicitin and a new protein 75 kDa elicitor were purified from the culture filtrate of Phytophthora palmivora, a pathogen of Hevea brasiliensis (rubber plant). Elicitin was obtained by using a one step of DEAE cellulose chromatography and the new elicitor was obtained by two steps of chromatography: a DEAE cellulose column followed by a hydrophobic column. Both elicitors were stable to heat and a wide range of pH values, but were sensitive to ProteaseK. Both elicitors induced scopoletin, peroxidase isozymes (with substrate o-dianisidine and scopoletin) and total phenolic compounds in cell suspension of H. brasiliensis with similar kinetics. In addition, both elicitors induced peroxidase enzyme (o-dianisidine), total phenolic compounds and enhanced local resistance against P. palmivora on young rubber tree seedlings. However, the increase of peroxidase enzyme and total phenolic compounds in rubber tree seedlings was different from those in cell suspension. Furthermore, during the expression of local resistance the zoospore of P. palmivora induced the peroxidase enzyme (o-dianisidine) more rapidly and with higher level than the control plants. H. brasiliensis is more responsive to the new elicitor than elicitin in triggering defense responses. That is the new elicitor was active at a concentration lower than those required for elicitin, about a 30-fold decrease for activation defense responses in cell suspension. For induction of peroxidase enzyme (o-dianisidine), phenolic compounds and local resistance of rubber plants against P. palmivora, the 75 kDa protein was active at about a 2-fold lower concentration when compared to elicitin.     

Fungi on roots and stem bases of asparagus in the Netherlands: species and pathogenicity

A survey was made to identify the most important soilborne fungal pathogens of asparagus crops in the Netherlands. Ten plants were selected from each of five fields with a young (1–4 y) first planting, five fields with an old (6–13 y) first planting and five fields with a young replanting. The analysis included fungi present in the stem base and the roots of plants with symptoms of foot and root rot or showing growth decline without specific disease symptoms. Isolates of each species were tested for pathogenicity to asparagus on aseptically grown plantlets on Knop's agar. Symptoms were caused byFusarium oxysporum, F. culmorum, Botrytis cinerea, Penicillium verrucosum var.cyclopium, Cylindrocarpon didymum, Phialophora malorum, Phoma terrestris andAcremonium strictum. F. oxysporum was by far the most common species and was isolated from 80% of the plants. Not all of its isolates were pathogenic to asparagus. Symptoms were caused by 67%, 78% and 93% of the isolates obtained from young first plantings, old first plantings and replantings, respectively.F. culmorum was isolated from 31% of the plants. Two other notorious pathogens of asparagus,F. moniliforme andF. proliferatum, did not occur in our samples.Species causing symptoms in the vitro test that were found on more than 5% of the plants were additionally tested for their pathogenicity in pot experiments.F. oxysporum f.sp.asparagi caused severe foot and root rot, significantly reduced root weights and killed most of the plants.F. culmorum caused lesions on the stem base often resulting in death of the plant.P. terrestris, a fungus only once reported as a pathogen of asparagus, caused an extensive root rot, mainly of secondary roots that became reddish. The fungus was isolated in only a few samples and is not to be regarded as an important pathogen in Dutch asparagus crops.P. malorum caused many small brown lesions on the stem base and incidentally also on the upper part of small main roots. This is the first report of its pathogenicity to asparagus. The fungus is one of the organisms inciting spear rust and it reduced crop quality rather than crop yield.P. verrucosum var.cyclopium andC. didymum did not cause symptoms in pot experiments.Because of its predominance on plants with foot and root rot and its high virulence,F. oxysporum f.sp.asparagi was considered to be the main soilborne pathogen of asparagus in the Netherlands.     

The oligopeptide elicitor Pep-13 induces salicylic acid-dependent and -independent defense reactions in potato

The Phytophthora-derived oligopeptide elicitor, Pep-13, originally identified as an inducer of plant defense in the nonhost–pathogen interaction of parsley and Phytophthora sojae, triggers defense responses in potato. In cultured potato cells, Pep-13 treatment results in an oxidative burst and activation of defense genes. Infiltration of Pep-13 into leaves of potato plants induces the accumulation of hydrogen peroxide, defense gene expression and the accumulation of jasmonic and salicylic acids. Derivatives of Pep-13 show similar elicitor activity in parsley and potato, suggesting a receptor-mediated induction of defense response in potato similar to that observed in parsley. However, unlike in parsley, infiltration of Pep-13 into leaves leads to the development of hypersensitive response-like cell death in potato. Interestingly, Pep-13-induced necrosis formation, hydrogen peroxide formation and accumulation of jasmonic acid, but not activation of a subset of defense genes, is dependent on salicylic acid, as shown by infiltration of Pep-13 into leaves of potato plants unable to accumulate salicylic acid. Thus, in a host plant of Phytophthora infestans, Pep-13 is able to elicit salicylic acid-dependent and -independent defense responses.     

Glomus intraradices, Pseudomonas alcaligenes, and Bacillus pumilus: effective agents for the control of root-rot disease complex of chickpea (Cicer arietinum L.)

The effects of Glomus intraradices, Pseudomonas alcaligenes and Bacillus pumilus on the root-rot disease complex caused by the root-knot nematode Meloidogyne incognita and the root-rot fungus Macrophomina phaseolina in chickpea was assessed by quantifying differences in the shoot dry mass, pod number, nodulation, and shoot content of chlorophyll, nitrogen, phosphorus and potassium. Inoculation of plants with G. intraradices, P. alcaligenes and B. pumilus alone and in combination significantly increased shoot dry mass, pod number, and content of chlorophyll, nitrogen, phosphorus and potassium in plants inoculated with pathogens over that in the uninoculated control plants. P. alcaligenes caused a greater increase in shoot dry mass, pod number, chlorophyll, nitrogen, phosphorus and potassium in plants with pathogens than did G. intraradices or B. pumilus. Combined application of G. intraradices, P. alcaligenes and B. pumilus to plants inoculated with pathogens caused a greater increase in shoot dry mass, pod number, nitrogen, phosphorus, and potassium than did an application of P. alcaligenes plus B. pumilus or of G. intraradices plus B. pumilus. Root colonization by G. intrardices was high when used alone, while inoculation with the pathogens reduced root colonization by G. intraradices. In the presence of P. alcaligenes and B. pumilus, root colonization by G. intraradices increased. In plants inoculated with just one antagonist, P. alcaligenes reduced galling and nematode multiplication the most, followed by G. intraradices, then B. pumilus. The greatest reduction in galling, nematode multiplication and root-rot was observed when both bacterial species and G. intraradices were applied together.     

褐飞虱与白背飞虱为害诱导水稻防御反应的比较

为探究2种稻飞虱——褐飞虱Nilaparvata lugens(St?l)和白背飞虱Sogatella furcifera(Horváth)诱导的水稻防御反应差异,于室内测定了水稻在分别受褐飞虱或白背飞虱产卵雌成虫为害时,其茉莉酸、水杨酸、乙烯、H_2O_2以及挥发物含量的变化。结果表明,尽管褐飞虱和白背飞虱产卵雌成虫的为害均可以诱导水稻茉莉酸、水杨酸、乙烯和H_2O_2等防御相关信号分子以及一些水稻挥发物含量的增加,但是二者的诱导作用存在差异。水稻在受白背飞虱产卵雌成虫为害时,茉莉酸的含量在3 h时就显著升高,12 h时含量达到最高;而受褐飞虱产卵雌成虫为害时,6 h时茉莉酸含量才显著升高,72 h时含量达最高;并且在2种稻飞虱为害的3～48 h内,白背飞虱为害诱导的茉莉酸含量始终显著高于褐飞虱为害诱导的。水稻受白背飞虱产卵雌成虫为害24 h后诱导的水杨酸含量、为害48 h后诱导的乙烯含量、为害72 h后诱导的H_2O_2含量及为害24 h后诱导的挥发物释放量分别是褐飞虱产卵雌成虫为害诱导的1.28、1.45、4.10和1.77倍。表明水稻能识别褐飞虱和白背飞虱的为害,从而做出针对害虫种类特异性的防御反应;并且水稻对白背飞虱产卵雌成虫为害所做出的防御反应比对褐飞虱的更强烈。     

Induction of systemic resistance,root colonisation and biocontrol activities of the rhizospheric strain of <Emphasis Type="Italic">Serratia plymuthica</Emphasis> are dependent on N-acyl homoserine lactones

Quorum sensing regulation, mediated by N-acyl homoserine lactone signals, produced by strain Serratia plymuthica HRO-C48 isolated from the rhizosphere of oilseed rape, was found to be responsible for this strain’s ability to produce the broad spectrum antibiotic pyrrolnitrin. In this study, we have shown that some other biocontrol-related traits of strain HRO-C48, such as protection of cucumbers against Pythium apahnidermatum damping-off disease, induced systemic resistance to Botrytis cinerea grey mold in bean and tomato plants, and that colonisation of the rhizosphere also depends on AHL signalling. The results prove that quorum sensing regulation may be generally involved in interactions between plant-associated bacteria, fungal pathogens and host plants.