Genetic regulation of conidiation in Trichoderma hamatum

Achieving a balance between vegetative growth and spore production is essential for successful biocontrol by fungi. Low sporulation rates in the field can result in poor establishment and survival, whereas failure of conidia to recognise hosts can lead to persistence without efficacy. Commercial biocontrol products involve bulk preparations of conidia, however considerable variability in conidiation rates exists between biocontrol agents, which can restrict choice of strain for production. The…     

The production relationship of destruxins and blastospores of Metarhizium anisopliae with virulence against Plutella xylostella

Metarhizium anisopliae as an essential entomopathogenic fungus has been known to produce destruxins(a kind of cyclo-peptidic mycotoxins) and blastospores in submerged culture. Blastospores and destruxins are candidates for insecticides, but the relations of both productions and the impact factors are unclear yet. In this study, we investigated the effects of inoculums, rotation, dissolved oxygen(DO) on the productions of blastospores and destruxins A and B(DA and DB) in submerged culture of M. anisopliae strain Ma Q01. The results indicated that DO levels were regulated by inoculum amounts and rotation speeds, meanwhile, the productions of DA, DB and blastospores were also closely influenced by those factors. Totally, when DO value was more than 40%, the higher productions of destruxins and blastospores were achieved, by contrast, lower than 40% of DO values resulted in lower productions. The regression analysis suggested that the productions of DA, DB and blastospores were positively correlated with the DO levels. Meanwhile, the positive correlations between the productions of DA or DB and blastospores were also found. Briefly, when the rotation is 150 r min–1 and the inoculum is 1.0×10~6 spore m L~(–1), the DA, DB and blastospores achieved the best production of 61.81 mg m L~(–1), 24.74 mg m L~(–1) and 5.73×10~6 spore m L~(–1), respectively. In addition, the pathogenicities of blastospores and conidia against Plutella xylostella were bioassayed. The higher mortalities of P. xylostella were totally recorded in blastospore treatments than in conidia treatments, especially in lower dosages and earlier periods. Our research will give some new insights to production of destruxins and blastospores by using M. anisopliae.     

Study on the character of chitinase produced by Trichoderna spp. with measuring reducing sugar

A trusty and intuitionistic method for screening chitinase produced by Trichoderma spp. was developed. 38 isolates of Trichoderma spp. were cultured in liquid medium with chitin or colloidal chitin as the sole carbon source for 4 days. The supernatant of the fermented broth was mixed with colloidal chitin and heated in water-bath at 37℃ for 30 min, then 3,5-dinitrosalicylic acid reagent (DNS) was added to the mixture, and let them react for 10 min in water-bath. According to the different colour of the mixture, the isolates of Trichoderma spp. which can produce chitinase could be screened.……     

培养条件对奶牛金葡菌5型荚膜多糖产量的影响（英文）

[Objective] The effect of different culture conditions on type 5 capsular polysaccharide production of Staphylococcus aureus from diary cattle was studied to provide simple way for CP production and preparation and laid foundation for carrying out new polysaccharide vaccine research.[Method] Staphylococcus aureus was isolated from milk sample of sick dairy cattle and capsular polysaccharide serotypes were identified.Type 5 capsular polysaccharide was cultured on BHI,solid columbia and mod110 culture media.Glucose and lactose were taken as carbon sources for every culture media in solid and liquid state.Therefore 9 different culture conditions were taken to study the effect of culture conditions on capsular polysaccharide production.[Result]Different culture conditions indicated that compared with columbia culture media,BHI culture media could decline capsular polysaccharide production and mod110 culture media could increase capsular polysaccharide production.While for same culture media,solid culture media was better for capsular polysaccharide production,meanwhile,taken lactose as carbon source could increase capsular polysaccharide production.     

Study on the character of chitinase produced by Trichoderma spp. with measuring reducing sugar

A trusty and intuitionistic method for screening chitinase produced by Trichoderma spp. was developed. 38 isolates of Trichoderma spp. were cultured in liquid medium with chitin or colloidal chitin as the sole carbon source for 4 days. The supernatant of the fermented broth was mixed with colloidal chitin and heated in water-bath at 37℃ for 30 min, then 3,5-dinitrosalicylic acid reagent (DNS) was added to the mixture, and let them react for 10 min in water-bath. According to the differe…     

Production of chitinases with Trichoderma harzianum isolates using solid substrate fermentation

Over forty Trichoderma harzianum isolates have been screened in solid substrate fermentation (SSF) for chitinase production. Strains were isolated from Asian soil and tree bark samples. Identification was performed in Canada and Austria by classical and molecular taxonomical methods. Four SSF media were used for the screening. They contained wheat bran, crude chitin from crab shells (SIGMA) and different salt solutions for wetting of the substrate. In a five day fermentation at 30…     

A new Curvularia lunata variety discovered in Huanghuaihai Region in China

The purpose of this study was to identify the dominant pathogens of Curvularia leaf spot and their pathogenicity variation in Huanghuaihai Region of China in recent years. In 2013 and 2016–2017, the occurrences of Curvularia leaf spots on maize were investigated in fields located in Henan, Hebei, Shandong, and Anhui provinces, and 292 fungi were isolated from diseased leaves. These fungal isolates were subjected to morphological identification, and 232 isolates were found to have about 70% uncurved conidia and were identified as Curvularia lunata var. Most of the conidia of 2 representative isolates, namely, HNWB-131 and HNWB-185, were oblong with parallel septations and were distinctly different from a reference isolate CX-3. For further determination, the internal transcribed spacer(ITS), glyceraldehyde 3-phosphate dehydrogenase(GPDH), the large subunit(LSU), and translation elongation factor 1-alpha(EF1-α) sequences of HNWB-131, HNWB-185, and CX-3 were amplified and sequenced. The results of sequence analysis showed that the 4 gene sequences from the 3 isolates had a similarity of more than 99% to C. lunata. Based on the sequences of ITS and the combined data of the 4 genes, neighbor-joining trees were constructed for phylogenetic analysis. The results indicated that these 3 isolates were clustered together with C. lunata. The expression of Clg2 p and ClUrase genes in mycelia and conidia was significantly(P0.05) higher in CX-3 than in HNWB-131 and HNWB-185. This study found that the dominant pathogen of Curvularia leaf spot was a new variety of C. lunata with morphological variations in Huanghuaihai Region from 2013 to 2017. The pathogenicity of the C. lunata var. was not significantly enhanced, and the expression of Clg2 p and ClUrase genes of C. lunata var. was decreased.     

Conidia of one Fusarium solani isolate from a soybean-production field enable to be virulent to soybean and make soybean seedlings wilted

Fusarium is usually thought to cause soybean root rot,which results in a large quantity of annual yield loss in soybean production,by its secretions including Fusarium toxins and cell wall degrading enzymes,but not by the conidia themselves that do not underlie any virulence so far.Here we report that the conidia of one Fusarium solani isolate are able to be virulent to soybean and make soybean seedlings wilted alone.We isolated them from the wilted plants in a soybean-production field and molecularly identified 17 Fusarium isolates through phylogenetic analysis.Of them,except for one isolate that showed diversity of virulence to different soybeans(virulent to one soybean whereas avirulent to another soybean),the others were all virulent to the two tested soybeans:both conidia cultures and secretions could make soybean seedlings wilted at 5 days post infection,and their virulence had dosage effects that only conidia cultures of at least 5×10~6 conidia mL~(-1) could show virulence to soybean;however,the sole conidia of the F.solani isolate #4 also exhibited virulence to soybean and could make soybean seedlings wilted.Finally,we developed the specific cleaved amplified polymorphic sequences(CAPS)markers to easily differentiate Fusarium isolates.The isolate #4 in this work will likely be used to investigate the new mechanism of virulence of Fusarium to soybean.     

Antagonism of local isolates of Trichoderna spp. on citrus root rot disease by Fusarium solani in the mekong delta of vietnam

The local isolates of Trichoderrma spp. and Fusariun solani were colected from citrus orchards in the Mekong delta of Vietnam and isolated on PDA, PDB and TSM medium for antagonism and Koch's postulate testing. The results showed that the high chitinolytic enzymes content of Trichoderma isolates can antagonise with Fusarium solani isolates by preventing the germination of Fusarium macroconidia in in-vitro condition. There are five promising isolates of Trichoderna spp. having high antagonism with Fusarium solani. These Trichoderma isolates also grew well in rice straws, maize stems, weeds and water hyacinth biowaste materials. These results supply the promising trend for biological control of root rot disease on citrus orchards of the Mekong delta.     

金龟子绿僵菌诱发UV-B抗性导致分生孢子产孢和孢子逆境耐受性的转化

The conidial tolerance of Metarhizium anisopliae var.anisopliae isolate ARSEF 2575 to UV-B irradiation is greatly influenced by growth-environment alterations. In this review, we report high variability in conidial UV-B tolerance in response to altered culture conditions.Conidia produced on insect cadavers[Zophobas morio(Coleoptera) or Galleria mellonella(Lepidoptera)] had low tolerance to UV-B radiation; and conidia produced on potato dextrose agar supplemented with yeast extract (PDAY) had medium UV-B tolerance; whereas conidia produced on a minimal medium without any carbon source (MM), on MM with a non-preferred carbon source such as lactose (=MML), on PDAY plus 1 M NaCl or KCl, or PDBY with high alkalinity had the highest UV-B tolerances. All of the above conditions that induced high UV-B tolerance, however, also greatly reduced conidial production. Comparisons between stress tolerance and conidial production, particularly with conidia produced under osmotic and nutritive stress, point out that the benefits of producing very tolerant conidia have the enormous cost of low conidial production. Growth under visible light also greatly improved conidial UV-B tolerance, but light did not negatively influence conidial production. Therefore, culture on rich media under light is proposed as the most promising approach to producing conidia with improved UV-B tolerance for biological control of pest insects in agriculture.     

Pathotype Structure of Magnaporthe grisea in the Fields of Chongyang and Yuan’an in Hubei Province,China

[Objective] This study aimed to explore the pathotype structure of Magnaporthe grisea in Chongyang and Yuan’an in Hubei Province,China.[Method] From the rice-growing fields of Chongyang and Yuan’an in Hubei Province where rice blast occurs frequently,60 isolates which were pathotyped against two sets of host differentials:Chinese host differentials and CO39 NILs,were obtained.Then,20 pathotypes with the six indica host differentials(CO39 NILs) were observed,while 13 pathotypes in four race groups were observed out of the same single spore isolates with Chinese host differentials which consists of three indica cultivars and four japonica cultivars.The diversity of the pathotypes of M.grisea populations tested by CO39 NILs was 2.54 and the pathotype 137.1 occurred at predominantly high frequency(21.67%).The diversity of physiological races of M.grisea populations tested by Chinese host differentials was 1.22 and the race group ZA occurred at predominantly high frequency(73.33%).The diversity of physiological races of M.grisea in Chongyang and Yuan’an were also calculated.Overall,the diversity of pathotypes of M.grisea in Yuan’an was higher than that in Chongyang with the two sets of the host differentials.[Conclusion] This study provided current information on the pathotype spectrum of M.grisea populations in the rice fields of Hubei Province to allow the formulation of viable strategies for blast resistance breeding programs in Hubei Province.     

Are mycoparasitism and chitinase production species or isolate dependent in Trichoderma ?

The relationship between taxonomic status of Trichoderma spp., chitinase production in solid substrate fermentation (SSF) on four media and mycoparasitism in dual culture (confrontation assay)against four plant pathogenic fungi was studied. Seventy five Trichoderma isolates belonging to 35species have been screened. The plant pathogenic fungi used in confrontation assay were Botrytis cinerea , Fusarium oxysporum f. sp. dianthi , Rhizoctonia solani and Sclerotinia sclerotiorum . The SSF media contained wheat bran, crude chitin (from crab shells, SIGMA) and salt solutions. The best performing isolates in mycoparasitism tests were Trichoderma flavofuscum, T. harzianum, T.inhamatum, T. koningii and T. strigosum. Some isolates exhibiting good mycoparasitism produced chitinase in SSF only at low or medium level. In contrary there were isolates with excellent extracellular chitinase production but their biocontrol potential did not belong to the leading group.Statistical methods have been used to evaluate the data.……     

美国西部重要害虫摩门螽斯病原真菌的研究

The Mormon cricket (MC),Anabrus simplex (Orthoptera:Tettigoniidae),has a long and negative history with agriculture in the western states of the USA where MC often migrates in large groups and causes significant damage to forage plants and cultivated crops.In this review, virulence to MC of isolates of the insect-pathogenic fungus Metarhizium anisopliae varieties acridum and anisopliae were compared in an effort to identify isolates with promise for use as MC biological control agents.All of the isolates tested induced 100% or nearly 100% mortality by six days post application of the fungal conidia.Searches for new Metarhizium isolates with high heat and UV-B tolerance included isolation fungi from field-caught MC and grasshopper after they died in the laboratory and culturing fungi from soil samples collected from numerous western USA sites.The survey was preceded by development of a dodine based selective medium that,at 0.002% active ingredient,permitted growth of M.anisopliae var.acridum,but inhibited most contaminating fungi.The M.anisopliae var.acridum isolates examined to date have much higher tolerance to heat and UV-B irradiation than M.anisopliae var.anisopliae isolates,and this may be critical to successful field applications.The variety acridum has not yet been found in the USA,so our search for such isolates continues.Several new M.anisopliae var.anisopliae and Beauveria spp.were found,and the Metarhizium isolates are being characterized as to stress tolerance and virulence to insects.Characterization includes comparisons of new and pre-existing Metarhizium isolates by amplified fragment-length polymorphism (AFLP) analysis.Finally, experiments on MC developmental biology were conducted to gather data needed to develop a degree day model and to establish laboratory colonies of MC.     

Sensitivities of Phytophthora infestans to Metalaxyl, Cymoxanil, and Dimethomorph

The isolates of Phytophthora infestans on tomato in the Guangxi Zhuang Autonomous Region, China, were determined for the sensitivities to metalaxyl, cymoxanil and dimethomorph to give the basic information for integrating disease management. Sensitivities were tested by measuring the radial growth on agar medium amended with fungicide, compared with the floating-leaf-disk method. 239 isolates were collected from eight tomato growing areas during 2000-2006. The testing results indicated that the frequencies of sensitive, intermediate, and resistant isolates to metalaxyl were 42.26, 35.98, and 23.53%, respectively. Variations in sensitivities amongst isolates from different areas or different years were very high for metalaxyl. All isolates from Tianlin and Wuxuan were sensitive to metalaxyl, but the metalaxyl-resistant isolates predominated in Tianyang, with the frequency of 51.35%. The EC50 values of certain isolates from Tianyang were higher than 500 μg·mL^-1 and their resistance levels were over 100 000 folds. Cymoxanil has been used for nearly 10 years in Guangxi, and dimethomorph has been used for 5-6 years. However, there was no decrease in sensitivity of P. infestans populations and the sensitivities of the pathogen were nearly normally distributed. Hence, their mean ECs0 value [cymoxanil （0.1647 ±0.0255） μg·mL^-1, dimethomorph （0.0970 ± 0.0052） μg·mL^-1 could be used as the baseline sensitivities for monitoring the field resistance development. The comparison with the floating-leaf-disk method indicates that both the techniques provided equivalent results. These studies suggested that metalaxyl can be continuously applied in Tianlin, Wuxuan, and Nanning due to the resistant isolates that have not been found, while for those areas with resistant isolate, the use of metalaxyl should be reduced or alternated, and cymoxanil or dimethomorph was recommended for controlling late blight disease of tomato.     

ISSR Marker and ITS Sequence Study of Melampsora Larici-populina

To compare the differences in intertranslation space of ribosomal DNA （ITS） of Melampsora larici-populina, between the isolates from China and isolates from other countries, this study investigated ITS sequences and ITS polygenetic tree based on 11 isolates that were collected from 5 races in different parts of China. The results indicated that there was no difference among the ITS sequences of 11 isolates from China. The ITS sequence of isolates from China was more homogeneous with that of isolates from Britain compared with France, Germany, and Canada. Intersimple sequence repeat （ISSR） markers were also used to study the genetic division of Melampsora larici-populina, and the results showed that the 11 tested isolates could be divided into Western population and Northern population. Genetic diversity index of race C2 was significantly different from that of races C4, C3, and C1, and no significant differences were observed among the other races. Pathogenicity division of races must not harmonize with their genetic division, except race C2. The ITS region is conservative, and ITS sequence is not fit for studying the differences that existed among the races. ISSR marker can be used for intraspecies population study, and Melampsora larici-populina in China can be divided into two populations.     

Physiological Characteristics of Boscalid Against Botrytis cinerea from Strawberry

A total of 96 monoconidial Botrytis cinerea strains were isolated from strawberry field.Physiological characteristics and resistance analysis to boscalid were investigated.Most isolates showed resistant phenotype and the resistance frequency of highly resistant isolates was as high as 45.10%.The effective concentration that inhibited conidia germination by 50% relative to the control values for sensitive isolates ranged from 0.068 to 60.56 μg· mL~(-1) in conidia germination assays.Compared to the sensitive strains, the boscalid-resistant strains had a significant increase in phenylalanine ammonia lyase(PAL) and peroxidase(POD) activity and more sensitivity to boscalid pressure.Fitness analysis showed statistically significant difference between sensitive and resistance isolates.Sequencing of Sdh B gene indicated that a single mutation corresponded to His272.The results suggested that resistance to boscalid in B.cinerea populations could result in failure to control gray mold.These results would enrich the understanding of the resistance mechanism of B.cinerea to boscalid.     

金龟子绿僵菌分生孢子暴露在B段紫外光下毒力的降低

The UV-A and UV-B wavelengths of solar radiation may seriously reduce viability of Metarhizium anisopliae conidia,a fungus used extensively in Brazil for insect pest control.The conidial tolerances to UV-A and UV-B radiation were studied for one isolate(IBCB 425) commercially available product produced on rice grains.Aqueous or oil emulsion conidial suspensions were irradiated for 0(control),2,or 3 h using the solar simulator filtered with diacetate filter to remove UV-C radiation which afforded 652 mW·m-2 in the spectral range of 280～370 nm.The UV tolerance was based on conidial germination(viability) and virulence to Diatraea saccharalis larvae.Conidia formulated with oil emulsion had higher survival after 3 h of UV exposure,but the virulence of the conidia previously exposed to 2 h of UV radiation were similar when oil formulated or not.The mortality of D.saccharalis caterpillars exposed to non-UV-irradiated conidia was greater than that of larvae exposed to irradiated conidia,and the LT50 values were lower for the non-irradiated suspensions than for those irradiated.This apparent reduction in virulence was directly related reduction in viability or delay of germination of UV-irradiated conidia.     

Effect of Different Bioassay Methods on Enzymatic Characteristics of Cotton Aphid,Aphis gossypfi Glover （Hemiptera：Aphididae）

In this study, the susceptibility of three populations of cotton aphid, Aph＆ gossypii Glover （Hemiptera：Aphididae） was assayed to imidacloprid （35SC） and thiametoxam （50WG）. The involvement of metabolic enzymes in the resistance strain of cotton aphid to the neonicotinoids was determined by the biochemical biomarkers and the resistance mechanism was determined as CaE. In another study, three different bioassay experiments were designed for detecting the susceptibility of cotton aphid to imidacloprid and thiametoxam and the effect of these two insecticides on the enzymatic activity of cotton aphid was assessed in the adult aphids treated with three different bioassay methods using a modified version of the FAO dip test, residue bioassay procedure and starvation method. Our findings suggested that the type of bioassay methods is very important when aphids＇ populations assess for the resistance against the neonicotinod insecticides. It has shown the starvation method is the most reliable method compared with other methods.     

Pathogenicity of Septoria carvi Syd. towards Caraway Carum carvi L, （Apiaceae）

Septoria carvi belong to the fungi recently detected on caraway plants in the Polish climatic conditions. In many European countries, this fungus causes significant losses in caraway cultivation and decreases the quality and quantity of the herb material. The aim of the present work was to study the fungus harmfulness towards the sowing materials, stems, leaves and umbels of caraway. The pathogenicity tests according to Koch＇s postulates were carried out for isolates ofS. carvi using post-culture liquids and a water suspension of conidia to inoculate the plants＇ parts. The studies were conducted in the laboratory, the climatic chamber and in field conditions. S. carvi caused disease symptoms in the form of spots and necrosis on all tested plants organs. The infection was followed by natural openings what was shown by scanning electron microscope （SEM） microscopic observations. The efficiency of inoculation increased injuries and anatomical dipping of plant epidermal tissue. The study showed the ability for asymptomatic S. carvi colonization of inoculated plants＇ parts. The performed pathogenicity tests and microscopic observations SEM showed that the tested isolates of S. carvi were occasional pathogens of caraway.     

Genetic regulation of conidiation in Trichoderma hamatun

Achieving a balance between vegetative growth and spore production is essential for successful biocontrol by fungi. Low sporulation rates in the field can result in poor establishment and survival,whereas failure of conidia to recognise hosts can lead to persistence without efficacy. Commercial biocontrol products involve bulk preparations of conidia, however considerable variability in conidiation rates exists between biocontrol agents, which can restrict choice of strain for production. The majority of studies on Trichoderma conidiation have focused on the species T. viride and T. atroviride.These species form conidia in response to blue and near-UV light and/or nutrient deprivation and conidiation proceeds in a highly co-ordinated fashion, however relatively little is known on the genetic basis of Trichoderrma conidiation. In addition, whilst photoconidiation appears to be a general response detailed studies in other Trichoderma species are absent. In this study, conidiation in the lesser known biocontrol species T. hamatum is being investigated using a combined morphological and molecular approach. In contrast to T. atroviride, conidiation in response to blue-light was weaker and variable and suggested that additional triggers may be required for the T. hamatum photoresponse. A series of comparative photoconidiation assays are currently being undertaken investigating the effect of inoculum type and abiotic factors on timing and intensity of the response.Results will be discussed in relation to the current knowledge on conidial morphogenesis in Trichoderma. In addition to these morphological assays, a selection of genes implicated in sporulation and the blue-light responses are currently being isolated and characterised from T. hamatum. Two genes, phr1 and cmp1 , which were isolated previously from T. atroviride will be used as early and late markers of gene expression during the photoresponse in T. hamatum in order to define time points for harvesting comparable stage-specific RNA from T. hamatum and T. atroviride. Using degenerate PCR putative sporulation gene orthologues have also been identified in T. hamatum.Work is currently underway to isolate genomic clones of these genes from T. hamatum and T.atroviride. Sequence and expression analysis of orthologues, including expression in response to abiotic factors will be presented and discussed in relation to the current knowledge of the molecular basis of conidiation in Trichoderma and other filamentous fungi.……