The genus Acrophialophora: History, phylogeny, morphology, beneficial effects and pathogenicity.

The genus Acrophialophora is a thermotolerant fungus, which is widely distributed in temperate and tropical zones. This fungus is classified in Ascomycota and belongs to the Chaetomiaceae family and the genera of Parathielavia, Pseudothielavia and Hyalosphaerella are closely related to Acrophialophora. For this genus have been reported 28 species so far, which two species of Acrophialophora jodhpurensis and Acrophialophora teleoafricana produce only sexual phase and other species produce asexual form. Therefore, producing both sexual and asexual forms were not reported by any species. Many applications were reported by some species in agriculture, pharmacy and industry. Production of enzymes, antimicrobial metabolites and plant growth-promoting factors were reported by some species. The species of A. nainiana is used in the industries of textile, fruit juice, pulp and paper due to extracellular enzyme production. Also, other species produce extracellular enzymes that can be used in various industries. The species Acrophialophora are used in the composting industry due to the production of various enzymes and to be thermotolerant. In addition, some species were isolated from hostile environmental conditions. Therefore has been suggested that it can be used for mycoremediation. Also, antimicrobial metabolites of Acrophialophora have been reported to be effective against human and plant pathogens. In contrast to the beneficial effects described, the Acrophialophora pathogenicity has been rarely reported. Two species A. fusispora and A. levis are opportunistic fungi and have been reported as pathogens in humans, animals and plants. Currently, the development and applications of Acrophialophora species have increased more than past. To our knowledge, there is no report with comprehensive information on the species of Acrophialophora, which include their disadvantage and beneficial effects, particularly in agriculture. Therefore, it seems necessary to pay more in-depth attention to the application of this genus as a beneficial fungus in agriculture, pharmaceutical and industry. This review is focused on the history, phylogeny, morphology, valuable roles of Acrophialophora and pathogenicity.

Efficacy of ergosterol peroxide obtained from the endophytic fungus Acrophialophora jodhpurensis against Rhizoctonia solani.

AIM: To investigate antifungal activity of the extract and major metabolite of the endophytic fungus Acrophialophora jodhpurensis (belonging to Chaetomiaceae) against crown and root rot caused by Rhizoctonia solani (teleomorph: Thanatephorus cucumeris), as an important pathogen of tomato. METHODS AND RESULTS: The endophytic fungus A. jodhpurensis, has high inhibitory effect against R. solani AG4-HG II in vitro and in vivo. The media conditions were optimized for production of the endophyte's metabolites. The highest amounts of secondary metabolites were produced at pH 7, 30°C temperature, and in the presence of 0.5% glucose, 0.033% sodium nitrate, and 1 gl-1 asparagine as the best carbon, nitrogen, and amino acid sources, respectively. The mycelia were extracted by methanol and the obtained extract was submitted to various chromatography techniques. Phytochemical analysis via thin-layer chromatography (TLC) and nuclear magnetic resonance (NMR) spectroscopy showed that ergosterol peroxide was the major component in the extract of this endophyte. Antifungal activities of the methanolic extract and ergosterol peroxide in the culture media were studied against R. solani. Minimum inhibitory concentrations of the extract and ergosterol peroxide against the pathogen were 600 and 150 µg ml-1, respectively. Ergosterol peroxide revealed destructive effects on the pathogen structures in microscopic analyses and induced sclerotia production. Histochemical analyses revealed that it induced apoptosis in the mycelia of R. solani via superoxide production and cell death. Application of ergosterol peroxide in the leaf disc assay reduced the disease severity in tomato leaves. CONCLUSIONS: Antifungal metabolites produced by A. jodhpurensis, such as ergosterol peroxide, are capable of controlling destructive Rhizoctonia diseases on tomato.

The Arac-like transcriptional regulator YqhC is involved in pathogenicity of Erwinia amylovora.

AIMS: This study aimed to identify virulence-associated genes and functions that affect disease development on pear caused by Erwinia amylovora EaUMG3 isolated from Iran. METHODS AND RESULTS: A mini-Tn5 transposon library was generated in EaUMG3. An E. amylovora mutant that had lost its ability to cause lesions on immature pear fruits, was selected for further analysis. This mutant was shown to have a transposon insertion in yqhC, a gene belongs to the AraC family of transcriptional regulators. A mutant of the wild-type EaUMG3 carrying an unmarked deletion of the yqhC gene was created using pDMS197. The Ea∆yqhC mutant showed reduced disease progression on immature pear fruits and pear plants, reduced motility and significantly lower levels of the virulence factors siderophore and amylovoran. Complementation with yqhC cloned in pBBR1MCS restored disease progression and the level of virulence factors to near wild type. CONCLUSION: YqhC transcriptional regulator is necessary for full virulence of E. amylovora. In addition, this regulator affects virulence factors such as siderophore production, amylovoran production, and motility. SIGNIFICANCE AND IMPACT OF STUDY: The identification of a novel transcriptional regulator with strong impact in the pathogenesis of E. amylovora, an organism causing significant economic losses in fruit production.

The Effect of Citrus Essential Oils and Their Constituents on Growth of Xanthomonas citri subsp. citri.

Citrus bacterial canker (CBC) caused by Xanthomonas citri subsp. citri (Xcc), is the most devastating of the citrus diseases worldwide. During our study, we found that Essential oils (EOs) of some citrus cultivars are effective on Xcc. Therefore, it prompted us to determine the plant metabolites responsible for the antibacterial properties. We obtained EOs from some locally cultivated citrus by using a Clevenger apparatus and their major constituents were identified by gas chromatography/mass spectrometry (GC-MS). The effect of Citrus aurantium, C. aurantifolia, Fortunella sp. EOs and their major constituents were evaluated against Xcc-KVXCC1 using a disk diffusion assay. Minimal inhibitory and bactericidal concentration of the EOs and their constituents were determined using the broth microdilution method. C. aurantium, C. aurantifolia Eos, and their major constituents including citral, linalool, citronellal, geraniol, α-terpineol, and linalyl acetate indicated antibacterial effects against Xcc. The C. aurantifolia EO and citral showed the highest antibacterial activity among the tested EOs and constituents with inhibition zones of 15 ± 0.33 mm and 16.67 ± 0.88 mm, respectively. Synergistic effects of the constituents were observed between α-terpineol-citral, citral-citronellal, citral-geraniol, and citronellal-geraniol by using a microdilution checkerboard assay. Transmission electron microscopy revealed that exposure of Xcc cells to citral caused cell wall damage and altered cytoplasmic density. We introduced C. aurantifolia and C. aurantium EOs, and their constituents citral, α-terpineol, citronellal, geraniol, and linalool as possible control agents for CBC.

Binucleate Rhizoctonia induced tomato resistance against Rhizoctonia solani via affecting antioxidants and cell wall reinforcement.

Isolates of Rhizoctonia solani (AG-3 PT, AG-4 HG-I, AG-4 HG-II) and one binucleate Rhizoctonia sp. (BNR) belonging to AG-Bb were investigated for pathogenicity on tomato cultivar Mobil. The BNR isolate revealed the lowest virulence and it was used as biocontrol agent against R. solani AG-4 HG-II, which showed the highest virulence on tomato. Inoculation of tomato plants with the hypovirulent BNR isolate reduced the disease symptoms of R. solani and induced resistance. Resistance induction was observed not only on the plants simultaneously inoculated with BNR and R. solani, but also when the plants were inoculated by the BNR and R. solani with time intervals. The peroxidase (POX), superoxide dismutase (SOD) and catalase (CAT) activities and expression levels of the corresponding genes in tomato plants increased after R. solani or BNR inoculation. The highest level of antioxidant activities and expression of their genes, lignin and callose formation were observed in the plants inoculated with the BNR and R. solani, simultaneously. The BNR inoculation reduced H2O2 accumulation. The highest level of priming was observed for the POX among other antioxidants tested via application of the BNR. Treatment with potassium cyanide (as a POX inhibitor) reduced basal resistance and BNR-induced resistance (BNR-IR) via reduction of lignification and callose deposition in tomato plants. These findings demonstrated the role of antioxidant enzymes, mainly the POX, in both basal resistance and BNR-IR. Therefore, redox state and antioxidants are involved in cell wall strengthening via lignin and callose formation, as important defense components which decrease the pathogen progress in plant tissues.

Genetic and Virulence Analysis of Rhizoctonia spp. Associated with Sugar Beet Root and Crown Rot in the Northeast Region of Iran.

Rhizoctonia spp. are the main causal agents of root and crown rot on sugar beet. In this study, isolates of Rhizoctonia spp. were obtained from diseased sugar beet in Iran over 2 years. Of 68 isolates, 61 were R. solani and 7 were R. cerealis. The anastomosis group (AG) of all isolates was determined on glass slides against the testers. Characterization of intraspecific groups (ISGs) of R. solani isolates revealed that, of 61 isolates, 43 were AG2-2 IIIB and 18 were AG2-2 IV. Amplified fragment length polymorphism (AFLP) analyses were used to investigate genetic structure of Rhizoctonia populations. Principal coordinate plots and cluster analysis differentiated R. solani from R. cerealis isolates and separated the R. solani isolates belonging to different ISGs. AFLP data indicated that the R. solani and R. cerealis populations are not clonal. Analysis of molecular variance in AG2-2 IIIB isolates showed that geographic region was the main factor determining genetic structure of the populations. Sampling year had no significant effect on the genotypes. Pathogenicity tests on Beta vulgaris 'FD0432' revealed that R. solani AG2-2 IIIB and AG2-2 IV isolates were more virulent than R. cerealis.

Crosstalk of nitro-oxidative stress and iron in plant immunity.

Accumulation of oxygen and nitrogen radicals and their derivatives, known as reactive oxygen species (ROS) and reactive nitrogen species (RNS), occurs throughout various phases of plant growth in association with biotic and abiotic stresses. One of the consequences of environmental stresses is disruption of homeostasis between production and scavenging of ROS and RNS, which leads to nitro-oxidative burst and affects other defense-related mechanisms, such as polyamines levels, phenolics, lignin and callose as defense components related to plant cell wall reinforcement. Although this subject has attracted huge interest, the cross-talk between these signaling molecules and iron, as a main metal element involved in the activity of various enzymes and numerous vital processes in the living cells, remains largely unexplored. Therefore, it seems necessary to pay more in depth attention to the mechanisms of plant resistance against various environmental stimuli for designing novel and effective plant protection strategies. This review is focused on advances in recent knowledge related to the role of ROS, RNS, and association of these signaling molecules with iron in plant immunity. Furthermore, the role of cell wall fortification as a main physical barrier involved in plant defense have been discussed in association with reactive species and iron ions.

Acrophialophora jodhpurensis: an endophytic plant growth promoting fungus with biocontrol effect against Alternaria alternata.

In this study, efficiency of the endophytic fungal isolate Msh5 was evaluated on promoting tomato plant growth and controlling Alternaria alternata, the causal agent of early blight in tomatoes. Morphological and molecular (ITS and tub2 sequences) analyses revealed that the fungal isolate, Msh5, was Acrophialophora jodhpurensis (Chaetomium jodhpurense Lodha). This beneficial fungus was capable of producing indole-3-acetic acid (IAA), urease, siderophore, extracellular enzymes, and solubilized phosphate. Under laboratory conditions, the Msh5 isolate of A. jodhpurensis inhibited A. alternata growth in dual culture, volatile and non-volatile metabolites assays. The supernatant of this endophytic fungus was capable of reducing spore germination and altering the hyphal structure of A. alternata and the spores produced germ tubes showed vacuolization and abnormal structure compared to the control. Also, the effect of A. jodhpurensis on plant growth parameters (such as shoot and root weight and length) and suppressing A. alternata was investigated in vivo via seed inoculation with spores of A. jodhpurensis using 1% sugar, 0.5% carboxymethyl cellulose (CMC) or 0.5% molasses solution as stickers. Colonization of tomato roots by the endophytic fungus resulted in significant increasing plant growth parameters and reduction in the progress of the diseases caused by A. alternata compared to the controls. Among the different coating materials used as stickers, sugar was found to be the most effective for enhancing plant growth parameters and decreasing the disease progress. Therefore, A. jodhpurensis isolate Msh5 can be suggested as a potential biofertilizer and biocontrol agent for protecting tomato plants against A. alternata.

Plant Growth-Promoting Activity of Pseudomonas aeruginosa FG106 and Its Ability to Act as a Biocontrol Agent against Potato, Tomato and Taro Pathogens.

P. aeruginosa strain FG106 was isolated from the rhizosphere of tomato plants and identified through morphological analysis, 16S rRNA gene sequencing, and whole-genome sequencing. In vitro and in vivo experiments demonstrated that this strain could control several pathogens on tomato, potato, taro, and strawberry. Volatile and non-volatile metabolites produced by the strain are known to adversely affect the tested pathogens. FG106 showed clear antagonism against Alternaria alternata, Botrytis cinerea, Clavibacter michiganensis subsp. michiganensis, Phytophthora colocasiae, P. infestans, Rhizoctonia solani, and Xanthomonas euvesicatoria pv. perforans. FG106 produced proteases and lipases while also inducing high phosphate solubilization, producing siderophores, ammonia, indole acetic acid (IAA), and hydrogen cyanide (HCN) and forming biofilms that promote plant growth and facilitate biocontrol. Genome mining approaches showed that this strain harbors genes related to biocontrol and growth promotion. These results suggest that this bacterial strain provides good protection against pathogens of several agriculturally important plants via direct and indirect modes of action and could thus be a valuable bio-control agent.

Farnesol altered morphogenesis and induced oxidative burst-related responses in Rhizoctonia solani AG1-IA.

Farnesol induces morphological changes characteristic of apoptosis in filamentous fungi. Growth-inhibitory effect and induced features of apoptosis on Rhizoctonia solani AG1-IA were observed in our study by addition of exogenous farnesol to the culture. The obtained results implied that farnesol triggered apoptosis-like features, such as production of reactive oxygen species (ROS), in R. solani AG1-IA and that there was increased superoxide dismutase (SOD) activity in the presence of farnesol, as well as decreased fungal biomass. Light microscopic analysis showed that farnesol disrupted the cytoplasm and deformed the hyphae of R. solani AG1-IA. The diameter of the hyphal cross-section in the fungus treated with farnesol decreased compared with control. Transmission electron microscopy (TEM) showed marked alternations in the cell wall, cell membrane, parenthesome, septum, and septal pore of the fungal cells. The findings of this work suggest that farnesol is deleterious to R. solani and has potential for use as an antifungal compound against this destructive phytopathogenic fungus.

Genomic, phylogenetic and catabolic re-assessment of the Pseudomonas putida clade supports the delineation of Pseudomonas alloputida sp. nov., Pseudomonas inefficax sp. nov., Pseudomonas persica sp. nov., and Pseudomonas shirazica sp. nov.

Bacteria of the Pseudomonas putida group are studied for a large panel of properties ranging from plant growth promotion and bioremediation to pathogenicity. To date, most of the classification of individual pseudomonads from this group relies on 16S RNA gene analysis, which is insufficient for accurate taxonomic characterization within bacterial species complexes of the Pseudomonas putida group. Here, a collection of 20 of these bacteria, isolated from various soils, was assessed via multi-locus sequence analysis of rpoD, gyrB and rrs genes. The 20 strains clustered in 7 different clades of the P. putida group. One strain per cluster was sequenced and results were compared to complete genome sequences of type strains of the P. putida group. Phylogenetic analyses, average nucleotide identity data and digital DNA hybridizations, combined to phenotypic characteristics, resulted in the proposition and description of four new species i.e. Pseudomonas alloputida Kh7 T (= LMG 29756 T = CFBP 8484 T) sp. nov., Pseudomonas inefficax JV551A3 T (= DSM108619 T = CFBP 8493 T) sp. nov., Pseudomonas persica RUB6 T (= LMG 29757 T = CFBP 8486 T) sp. nov. and Pseudomonas shirazica VM14 T (= LMG 29953 T = CFBP 8487 T) sp. nov.

Characterization, Genetic Structure, and Pathogenicity of Rhizoctonia spp. Associated with Rice Sheath Diseases in India.

ABSTRACT Isolates of Rhizoctonia spp. were obtained from rice in India during 2000-2003. Characterization by conventional techniques and polymerase chain reaction showed that from 110 isolates, 99 were R. solani and 11 were R. oryzae-sativae. Of 99 isolates identified as R. solani, 96 were AG1-IA, 1 was AG1-IB, and 2 were AG1-IC. Amplified fragment length polymorphism (AFLP) analyzes were used to determine genetic relationships in Rhizoctonia pathogen populations collected from different geographic regions. Cluster analysis based on the AFLP data separated isolates belonging to the three different intraspecific groups of R. solani AG1 and differentiated R. solani from R. oryzae-sativae. Analysis of molecular variance (AMOVA) revealed that geographic region was the dominant factor determining population structure of R. solani AG1-1A; host cultivar had no significant effect. Pathogenicity tests on Oryza sativa cv. Zenith revealed that isolates of R. solani AG1-1A and AG1-1B were more virulent than R. solani AG1-IC and R. oryzae-sativae isolates.

Reactive oxygen species accumulation and homeostasis are involved in plant immunity to an opportunistic fungal pathogen.

Alternaria blight is a major and destructive disease of potato worldwide. In recent years, A. tenuissima is recognized as the most prevalent species of this phytopathogenic fungus in potato fields of Asian countries, which causes high yield losses every year. Any potato cultivar with complete resistance to this disease is not recognized, so far. Therefore, screening resistance levels of potatoes and identification of plant defense mechanisms against this fungus might be important for designing novel and effective disease management strategies for controlling the disease. In this research, the role of reactive oxygen species, antioxidants, lignin and phenolics in potato basal resistance to A. tenuissima was compared in the partially resistant Ramus and susceptible Bamba cultivars. Priming O2- and H2O2 production and enhanced activity of peroxidase (POX) and catalase (CAT) during interaction with A. tenuissima were observed in Ramus cultivar. Application of ROS generating systems and scavengers revealed critical role of O2- and H2O2 in potato defense, which was associated with lignification and phenolics production. More OH- and lipid peroxidation in the susceptible Bamba compared to Ramus cultivar showed their negative effects on resistance. Priming the POX and CAT activity, in correlation with upregulation of the corresponding genes was observed in Ramus. The POX and CAT inhibitors increased disease progress, which was related with decreased lignification. This assay demonstrated not only POX-dependency of lignification, but also its dependence on CAT. However, POX had more importance than CAT in potato defense and in lignification. These findings highlight the function of ROS accumulation and homeostasis in potato resistance against A. tenuissima.

Phylogenetic diversity and antagonistic traits of root and rhizosphere pseudomonads of bean from Iran for controlling Rhizoctonia solani.

Fluorescent pseudomonads from bean root and rhizosphere in Iran were investigated for biocontrol of the fungal pathogen Rhizoctonia solani. Phylogenetic analysis of concatenated 16S rRNA, gyrB and rpoD sequences for 33 Pseudomonas isolates showed that 15 belonged to four clusters within the 'P. fluorescens' group, i.e. one corresponding to P. thivervalensis, two others including P. moraviensis or P. baetica, and the last one without closely-related established species. The 18 other isolates belonged to five clusters within the 'P. putida' group, one including P. mosselii and P. entomophila, another including strains currently described as P. putida, and three without closely-related species described. Ten isolates were selected based on in vitro inhibition of R. solani. Cellulase activity was identified in three pseudomonads, chitinase activity in two pseudomonads, extracellular protease activity in nine pseudomonads and hydrogen cyanide production in two pseudomonads. Genes coding for production of phenazine, pyoluteorin, pyrrolnitrin and 2,4-diacetylphloroglucinol were not found, whereas the 1-aminocyclopropane-1-carboxylate deamination gene acdS was present in three pseudomonads. The antagonistic acdS+ strain VKh13 from the 'P. putida' group effectively protected soil-grown bean from R. solani AG 4-HGI. Results show that pseudomonads from uncharacterized taxa were readily obtained from Iranian soils and displayed biocontrol potential against R. solani.

Whole-Genome Sequence of Pseudomonas fluorescens EK007-RG4, a Promising Biocontrol Agent against a Broad Range of Bacteria, Including the Fire Blight Bacterium Erwinia amylovora.

Here, we report the first draft whole-genome sequence of Pseudomonas fluorescens strain EK007-RG4, which was isolated from the phylloplane of a pear tree. P. fluorescens EK007-RG4 displays strong antagonism against Erwinia amylovora, the causal agent for fire blight disease, in addition to several other pathogenic and non-pathogenic bacteria.

The role of nitric oxide in basal and induced resistance in relation with hydrogen peroxide and antioxidant enzymes.

Nitric oxide (NO) is one of the main signal molecules, which is involved in plant growth and development and can change regular physiological activity in biotic and abiotic stresses. In this study, the role of NO in induced resistance with Pseudomonas fluorescent (CHA0) and basal resistance against Rhizoctonia solani in bean plant was investigated. Our results revealed that P. fluorescent and R. solani can increase NO production at 6h post inoculation (hpi). Also, using the NO donor S-nitroso-N-acetyl D-penicillamine (SNAP) led to increase NO and bean plant resistance against R. solani. Utilizing the NO scavenger, 2-(4-carboxyphenyl)-4,4,5,5-tetramethy-limidazoline-1-oxyl-3-oxide (cPTIO), not only decreased basal resistance but also reduced induced resistance. In continue, the activity of antioxidant enzymes was studied in the former treatments. SNAP, CHA0 and R. solani increased the activity of peroxidase (POX), catalase (CAT) and ascorbate peroxidase (APX) at 6, 12 and 24h post inoculation (hpi). In contrast, using cPTIO and R. solani simultaneously (cPTIO+R) showed reduction in activity of POX and APX at 6 hpi. The cPTIO+R treatment increased POX, APX and CAT activity at 12 and 24 hpi. Hydrogen peroxide (H2O2) monitoring in the leaf discs clarified that SNAP can increase H2O2 production like CHA0 and R. solani. On the other hand, SNAP increased the resistance level of leaf discs against R. solani. Treating the leaf discs with cPTIO led to decrease resistance against the pathogen. These leaf discs showed reduction in H2O2 production at 6 hpi and suddenly enhanced H2O2 generation was observed at 24hpi. This study showed that CHA0 can increase NO level in bean plants. NO induced H2O2 generation and regulated redox state of the host plant. This interaction resulted in significant defense against the pathogen.

A survey on basal resistance and riboflavin-induced defense responses of sugar beet against Rhizoctonia solani.

We examined basal defense responses and cytomolecular aspects of riboflavin-induced resistance (IR) in sugar beet-Rhizoctonia solani pathsystem by investigating H(2)O(2) burst, phenolics accumulation and analyzing the expression of phenylalanine ammonia-lyase (PAL) and peroxidase (cprx1) genes. Riboflavin was capable of priming plant defense responses via timely induction of H(2)O(2) production and phenolics accumulation. A correlation was found between induction of resistance by riboflavin and upregulation of PAL and cprx1 which are involved in phenylpropanoid signaling and phenolics metabolism. Application of peroxidase and PAL inhibitors suppressed not only basal resistance, but also riboflavin-IR of sugar beet to the pathogen. Treatment of the leaves with each inhibitor alone or together with riboflavin reduced phenolics accumulation which was correlated with higher level of disease progress. Together, these results demonstrate the indispensability of rapid H(2)O(2) accumulation, phenylpropanoid pathway and phenolics metabolism in basal defense and riboflavin-IR of sugar beet against R. solani.