Cloning of cDNA Fragment of Chitinase Gene from the Mycoparasite Trichoderma atroviride on Armandii Pine Blister Rust

[Objective] The aim of this study was to isolate chitinase gene from Trichoderma atroviride strain SS003. [Method] With the aeciospore wall of armandii pine blister rust as inducer, chitinase gene was induced to express in Trichoderma atroviride cells. The cDNA fragment of chitinase gene was cloned by RT-PCR approach. [Result] The activity of chitinase induced reached 40.17 μg/10 min; and the specific fragment amplified was 834 bp in length and proved to be the fragment of chitinase gene by sequencing and sequence analysis. [Conclusion] The result showed the feasibility of isolating the full length of chitinase gene and its transformation, and further producing chitinase.

Antifungal Potential of Beauveria bassiana on Solanum lycopersicum L. Infected with Fusarium oxysporum f. sp. lycopersici

The objective of this work was to evaluate the effect of Beauveria bassiana(Bb 1205)on controlling Fusarium oxysporum f.sp.lycopersici(Fol 17108)in tomato plants in greenhouse conditions.Inoculation of Bb 1205 was the most promising among the agronomic variables and expression of the activity of the enzymesβ-1,3-glucanases and chitinases.Inoculation of Bb 1205 occurred at a concentration of 1×108 conidia·mL−1,which was administered onto the leaves,directly into the soil and via injection.Infection with Fol 17108 occurred with 1×106 spores·mL−1,which were added directly to the soil.Spectrophotometry was used for measuring agronomic parameters,namely activity of chitinases andβ-1,3-glucanases in foliage and roots.When Bb 1205 was added to the soil,the chlorophyll index and aerial part length showed significant differences.In addition,it was determined that root length,fresh weight of foliage,flower,and fruit count increased 82 days after inoculation(dai).Chitinase activity induced by Bb 1205 in leaves and roots of tomato plants infected with Fol 17108 was observed when injected into the stem at 32 dai(41.8 and 11.6-fold,respectively).Inoculation on the foliage showed a 10-fold increase ofβ-1,3-glucanases in the roots after 82 dpi.As for leaves,a 3.8-fold increase was found when the stem was inoculated.In the different in vivo applications,Bb 1205 activated its defenses by expressing the chitinase enzymes andβ-1,3-glucanase,thus reducing the damage caused by Fol 17108,demonstrating increase plant growth thereafter.

Mycoparasitism of Nematode-Trapping Fungus Monacrosporium ellipsosporumand Its Biochemical Basis

Monacrosporium ellipsosporum, a nematode-trapping fungus, was isolated by baiting with sclerotiaof Sclerotinia sclerotiorum in soil from a tobacco field in Yuxi, Yunnan Province. Colonizationfrequency of the sclerotia by the fungus was 18% in natural soil. Reinoculation tests byplacing surface-sterilized sclerotia on fungal cultures for two weeks and then surface-sterilized again led to 32% sclerotia be infected. Dual culture tests in PDA plates did notgive rise to a suppression zone between the colonies of M. ellipsosporum and its counterpartfungi S. sclerotiorum and Rhizoctonia solani, suggesting there was little or no nutritionalcompetition and absent of antifungal compounds. However, M. ellipsosporum could grow overabsent of S. sclerotiorum and R. solani, and significantly inhibited their growth on agarplates. Scanning electron and light microscopic observations showed that hyphae of M. ellipsosporumgrew along and appressed on hypha of S. sclerotiorum and coiled around hyphae of R. solani.Assays of cell wall-degrading enzymes showed that M. ellipsosporum grew well in chitin agarmedia, with clear transparent hydrolysis zones. Activities of total chitinase, exo-chitinase,β-1, 3-glucanase and protease were 140.2±11.9, 82.9±4.1, 111.2±7.6 and 76.1±4.3 U respect-ively, after incubation for 4 days at 30 ℃ in liquid media containing ground sclerotia of S.sclerotiorum as sole nutrient source. These enzymes might be important in the mycoparasiticactivity of M. ellipsosporum.

Evaluating effective Trichoderma isolates for biocontrol of Rhizoctonia solani causing root rot of Vigna unguiculata

The highly diverse genus Trichoderma has provided many formulations that are alternatives to the chemical pesticides in agriculture.The present study was undertaken to investigate the biocontrol potential of eight Trichoderma species,T.atrobrunneum,T.guizhouense,T.paratroviride,T.pyramidale,T.rufobrunneum,T.simmonsii,T.thermophilum and T.viridulum,against the phytopathogenic fungus Rhizoctonia solani.Trichoderma isolates were first evaluated in vitro by dual culture tests for their antagonism,mycoparasitic ability and antifungal activity against R.solani.Their growth promoting potential was further assessed in relation to phosphate solubilization,indole acetic acid and siderophore production.Five of the isolates were selected and evaluated for their abilities to prompt plant growth and to control R.solani infecting Vigna unguiculata(cowpea)seedlings in vivo.Two most effective isolates,T.guizhouense 9185 and T.simmonsii 8702,significantly(P<0.05)reduced the disease severity incidences(36.6 and 45.0%,respectively)and promoted plant growth,which have good prospects for application.

Laboratory efficacy of mycoparasitic fungi(Aspergillus tubingensis and Trichoderma harzianum) against tropical bed bugs(Cimex hemipterus)(Hemiptera:Cimicidae)

Objective: To test the effectiveness of conidial spore formulations [Aspergillus tubingensis(A.tubingensis) and Trichoderma harzianum(T.harzianum)] against tropical bed bugs, Cimex hemipterus.Methods: Spore formulations were made from two fungal strains, T.harzianum and A.tubingensis.The bed bugs were exposed to the conidial spores placed soaked onto a fabric cloth for 1 h and the mortality counts were recorded daily until 14 days.Results: Mean survival times based on Kaplan–Meier survival analysis showed no significant differences between all the concentrations in both the fungal isolates:T.harzianum and A.tubingensis.However, the evaluation of both the isolates in terms of virulence resulted in low lethal hours in all the concentrations except for the high concentration of A.tubingensis(LT_(50)= 44.629 h) at the conidial exposure of 1 × 10~6 spores/mL.Rapid mortality of the bed bugs was observed from Day 6 to Day 12, ranging from 13% to 90% in all three concentrations of A.tubingensis.With reference to the T.harzianum exposure, the concentration of 1 × 10~4 spores/mL displayed a gradual increase in the percentage mortality of 90 on Day 14.Conclusions: Approaches to the bed bugs treatment should be explored in-depth using a natural biological agent like fungus especially A.tubingensis to reduce this pest population, in order to replace chemical methods.

Factors that contribute to the mycoparasitism stimulus in Trichoderma atroviride. strain P1

Trichoderma atroviride strain P1 has been used extensively to study the mycoparasitic mechanisms in the interaction between plant pathogenic host and beneficial antagonistic fungi. Mutants of P1 containing the green fluorescent protein (gfp) or glucose oxidase (gox) reporter systems and different inducible promoters (from the exochitinase nag1 gene, or the endochitinase ech42 gene of P1) were used to determine the factors that activate the biocontrol gene expression cascade in the antagonist. The following compounds were tested singly and in various combinations: purified Trichoderma P1 enzymes (endochitinase, exochitinase, chitobiosidase, glucanase); antagonist culture filtrates (T. atroviride P1 wild-type and relative knock-out mutants, T. harzianum, T. reesei); pathogen culture filtrates (Botrytis, Pythium, Rhizoctonia); purified fungal cell walls (CWs) from Trichoderma, Botrytis, Pythium, Rhizoctonia; colloidal crab shell chitin; and plant extracts from cucumber leaves, stems or roots. Strong induction of mycoparasitism was found with the various digestion products produced by treating fungal CWs and colloidal chitin with purified enzymes or fungal culture filtrates. Filtrates from chitinase knock-out mutants, as well as CWs from Oomycetes fungi, were less active in producing the stimulus for mycoparasitism. The host CW digestion products were separated by molecular weight (MW) to determine which compounds were able to activate Trichoderma. Micromolecules of MW less than 3 kDa were found to trigger mycoparasitism gene expression before physical contact with the host pathogen. These compounds stimulated mycelial growth and spore germination of the antagonist. Purification of these host-derived compounds was conducted by HPLC and in vivo assay. The obtained inducers were able to stimulate both the production of endochitinase and exochitinase enzymes, even under repressing conditions in the presence of glucose. Inducers stimulated the biocontrol effect of P1 in the presence of host fungi. The disease symptom development on bean leaves inoculated with Botrytis and Trichoderma spores was clearly reduced by the addition of the inducers, unless these molecules were not specifically inactivated. Finally, purified inducers added to liquid cultures of T. atroviride P1 stimulated the production of low MW antibiotics and metabolites which inhibited Botrytis spore germination. Mass spectrometry analysis (ESI-MS) of the inducers indicated the presence of hexose oligomers, like cellobiose, while MS/MS analysis by selective fragmentation of peaks in the spectrum demonstrated the presence of at least three distinct compounds that were biologically active.

Fungicolous fungi:terminology,diversity,distribution,evolution,and species checklist

Fungicolous fungi are a very large,diverse,ecological and trophic group of organisms that are associated with other fungi.This association occurs with species of different lineages across the fungal kingdom.They are recognized as symbionts,mycoparasites,saprotrophs,and even neutrals.Wherever fungi have been found,fungicolous taxa have also been found.Homogeneous environments favour the development of highly adapted and coevolved fungicolous species,which could have led to host-specificity aspects.As a primary consumer,fungicolous fungi decrease the turnaround time of certain nutrients in food webs,due to their special often-rapid life cycles.They may also significantly affect population dynamics and population sizes of their hosts in aquatic or terrestrial ecosystems.As mycoparasites of pathogenic fungi,some fungicolous fungi have been explored as biocontrol agents.They may also cause serious diseases of cultivated edible and medicinal mushrooms,decreasing both yield and quality.Fungicolous fungi could be used as model organisms that may help determine better understanding of species interactions,fungal evolution and divergence,and fungicolous mechanisms.This review summarizes our current understanding of fungicolous fungi,with a particular focus on the terminology,diversity,global distribution,and interaction with their hosts.We also provide a checklist including 1552 fungicolous fungal taxa so far recorded following the updated classification schemes.There is a need for further investigations on this ecologically important group of fungi to better understand their biology,ecological aspects,origin and divergence,hostspecificity and application in biocontrol.Accurate identification of these fungi as pathogens and their significance in quarantine purposes on the mushroom industry need further evaluations so that efficient control measures can be developed for better disease management purposes.

The genus Pythium in Taiwan,China(1)-a synoptic review

The genus Pythium,with slightly over 280 described species,has been classified traditionally with other filamentous,coenocytic,sporangia-producing fungi as“Phycomyetes”.However,with recent advances in chemical,ultrastructural and molecular studies,Pythium spp.are now considered as“fungus-like organisms”or“pseudo-fungi”and are placed in the Kingdom Chromista or Kingdom Straminopila,distinct from the true fungi of the Kingdom Fungi or Kingdom Mycota.They are widely distributed throughout the world as soil saprophytes or plant pathogens.Because of the warm moist maritime climate,Taiwan,China,is especially rich in Pythium species.To date,48 species of Pythium have been reported from Taiwan,China,with the dominant species being Py.vexans,Py.spinosum,Py.splendens,Py.aphanidermatum,Py.dissotocum and Py.acanthicum.There is no definite geographical distribution of Pythium spp.in Taiwan,China.Twenty nine species of Pythium have proven to be plant pathogens attacking a wide variety of woody and herbaceous plants primarily causing pre-and post-emergence seedling damping-off,root rot,stem rot and rotting of fruits,tubers and ginger rhizomes,resulting in serious economic losses.The most important plant pathogenic species include Py.aphanidermatum and Py.Myriotylum,which are most active during the hot and wet summer months;whereas Py.splendens,Py.spinosum,Py.ultimum and Py.irregulare cause the greatest damage in the cool winter.Most Pythium spp.are non-specific pathogens,infecting mainly juvenile or succulent tissues.This review attempts to assess the taxonomic position of the genus Pythium and provide details of the historical development of the study of Pythium as pathogens in Taiwan,China,causing diseases of sugarcane,trees,vegetables,fruits,specialty crops and flowering plants,as well as measures to control these diseases.Of special note is the introduction of the S-H mixture which,when used as soil amendment,effectively controls many soil-borne Pythium diseases during the early stages of plant growth.The diversity of Pythium species in Taiwan,China,is discussed in comparison with the situation in the mainland of China and suggestions are made to fully utilize Pythium spp.as agents for biological control,in industry and medicine.

Deletion of all three MAP kinase genes results in severe defects in stress responses and pathogenesis in Fusarium graminearum

Mitogen-activated protein kinase(MAPK)cascades are activated by external stimuli and convert signals to cellular changes.Individual MAPKs have been characterized in a number of plant pathogenic fungi for their roles in pathogenesis and responses to biotic or abiotic stresses.However,mutants deleted of all the MAPK genes have not been reported in filamentous fungi.To determine the MAPK-less effects in a fungal pathogen,in this study we generated and characterized mutants deleted of all three MAPK genes in the wheat scab fungus Fusarium graminearum.The Gpmk1 mgv1 Fghog1 triple mutants had severe growth defects and was non-pathogenic.It was defective in infection cushion formation and DON production.Conidiation was reduced in the triple mutant,which often produced elongated conidia with more septa than the wild-type conidia.The triple mutant was blocked in sexual reproduction due to the loss of female fertility.Lack of any MAPKs resulted in an increased sensitivity to various abiotic stress including cell wall,osmotic,oxidative stresses,and phytoalexins,which are likely related to the defects of the triple mutant in environmental adaptation and plant infection.The triple mutant also had increased sensitivity to the biocontrol bacterium Bacillus velezensis and fungus Clonostachys rosea.In co-incubation assays with B.velezensis,the Gpmk1 mgv1 Fghog1 mutant had more severe growth limitation than the wild type and was defective in conidium germination and germ tube growth.In confrontation assays,the triple mutant was defective in defending against mycoparasitic activities of C.rosea and the latter could grow over the mutant but not wild-type F.graminearum.RNA-seq and metabolomics analyses showed that the MAPK triple mutant was altered in the expression of many ATP-binding cassette(ABC)and major facilitator superfamily(MFS)transporter genes and the accumulation of metabolites related to arachidonic acid,linoleic acid,and alpha-linolenic acid metabolisms.Overall,as the first study on mutants deleted of all three MAPKs in fungal pathogens,our results showed that although MAPKs are not essential for growth and asexual reproduction,the Gpmk1 mgv1 Fghog1 triple mutant was blocked in plant infection and sexual reproductions.It also had severe defects in responses to various abiotic stresses and bacterial-or fungal-fungal interactions.

Stem Blight Control of Schizonepeta tenuifolia Caused by Phytophthora nicotianae Using Trichoderma spp.

Objective To control stem blight disease of Schizonepeta tenuifolia caused by Phytophthora nicotianae. Methods The antagonist effect of 13 Trichoderma strains (including T. viride and T. harzianum) was evaluated upon mycelia growth of P. nicotianae. Trichoderma strains with high antagonistic activities against the pathogen were used to control stem blight of S. tenuifolia in the field. Results Of 13 Trichoderma strains tested, T. viride strain M3 showed maximum mycelia growth inhibition (83.2%) to the pathogen, followed by T. viride strain Tv04-2 (78.2%) and then T. harzianum strain ThB (65.0%), in vitro. Fungal cell wall degrading enzymes, protease, and β-1,3-glucanase were analyzed qualitatively and quantitatively in further study. T. viride strains M3, Tv04-2, and T. harzianum strain ThB efficiently against P. nicotianae were used to control stem blight of S. tenuifolia in the field, and T. viride strain M3 showed the best biocontrol potential. Conclusion Trichoderma spp. can be used as alternatives of pesticides to control stem blight, one of the serious soilborne diseases of S. tenuifolia caused by P. nicotianae. However, though T. viride strains Tv04-2 and T. harzianum strain ThB are also highly against P. nicotianae in vitro, the controlling efficacy of them on stem blight disease is not as excellent as T. viride strains M3 in the field.