Optimization of Sporulation Conditions of Biocontrol Bacteria B579 by Two-step Control Strategy

[Objective] For preparing the biopesticide of Bacillus subtilis B579 with high spore concentration,the sporulation conditions were optimized.[Method] Two-step fermentation control strategy was used,in which,the first phase（0-10 h）was to improve cell growth,and the second phase（10-30 h）was to promote spore formation.Four factors including initial glucose concentration,fermentation pH,temperature（in the second phase）and shaking speed（in the second phase）were optimized using the methods of single factor test and orthogonal experiment.[Result] The initial glucose concentration showed a significant effect on sporulation.The optimal conditions for the spore formation of B.subtilis B579 were as follows：initial glucose concentration 5 g/L,fermentation pH 7.0,the temperature for the first phase 37 ℃,and the shaking speed for the first phase 180 r/min,the temperature for the second phase 40 ℃,and the shaking speed for the second phase 200 r/min;in addition,the first phase was 10 h and the second phase of fermentation was conducted for 30 h.Under such conditions,the spore concentration and spore formation rate could reach 9.43×108 CFU/ml and 90.99%,respectively,which represented 6.70-fold and 2.43-fold increase compared with those before optimization.[Conclusion] The spores concentration of biocontrol agent was improved using two-step control strategy,which provided the basis for biopesticide production in large scale.

Improvement of Sporulation Conditions of a New Strain of Bacillus amyloliquefaciens in Liquid Fermentation

The desirable active ingredients for the development of bioproducts based in Bacillus sp. for the control of soil pathogens are the spores because these structures exhibit more resistance and stability to conditions present during the fermentation, formulation, and storage processes. To improve the sporulation of a native strain of Bacillus amyloliquefaciens (Bs006) using liquid fermentation, some modifications in the concentrations of the components in a previously standardized culture media were made. Subsequently, five sporulation inducers (iron nitrate, mixture of salts, peroxide hydrogen, temperature, and initial cell concentration) were evaluated. The treatment with a mixture of salts in combination with an initial cell concentration of 1 × 108 cells/ml was selected because a final spore concentration of 1.05 × 1010 spores/ml after 66 hours with a fully substrate consumption and sporulation efficiency of 88.7% was obtained. To demonstrate the biological activity of B. amyloliquefaciens Bs006 in Cape gooseberry seedlings, a greenhouse bioassay was conducted, and statistical differences in plant growth-promoting parameters compared with previous media were not found. Additionally, the proposed modified media (coded as JM) presented a benefit-cost ratio 2.65 times higher compared with the baseline media.

Biological Characteristics of the Pathogen Phyllosticta capitalensis Causing Banana Freckle Disease in Hainan Province

[Objectives]This study was conducted to clarify the biological characteristics of the pathogen Phyllosticta capitalensis,the causal agent of freckle disease on Cavendish banana in Hainan Province,China.[Methods]The impact of various nutritional and environmental factors,including media,carbon sources,nitrogen sources,temperature,pH and light on the growth and sporulation of P.capitalensis was assessed using two distinct methods:mycelium growth rate and blood counting chamber.[Results]The mycelial growth and sporulation of P.capitalensis on different media exhibited notable differences.The use of banana leaf extract dextrose agar(BLEAD)and carrot agar(CA)was observed to facilitate rapid mycelial growth.The potato dextrose agar(PDA)and potato sucrose agar(PSA)were conducive to the production of conidia.The utilization of distinct carbon and nitrogen sources exerted a pronounced influence on the growth of P.capitalensis.Maltose,dextrose,fructose,and casein acid hydrolysate were the preferred substrates for mycelial growth.The tested carbon and nitrogen sources did not significantly stimulate conidial production,whereas dextrose and NaNO 3 were found to favor sporulation.The optimal temperature for mycelial growth and conidial production was determined to be 28 and 32℃,respectively.No mycelial growth was observed at 5℃.Active mycelial growth was observed at pH 6-10,with pH 6-7 being particularly conducive to sporulation.Complete darkness was conducive to mycelial growth and sporulation.[Conclusions]It is recommended that BLEDA and PDA should be incubated at 28℃for 14 d in the dark for the purpose of mycelial growth and sporulation of P.capitalensis,respectively.

Influence of Long-Term Fixed Fertilization on Diversity of Arbuscular Mycorrhizal Fungi

Diversity of arbuscular mycorrhizal fungi(AMF) was investigated in a field that had received long-term fixed ferti-lization(LFF) for 26 years.There were a total of 12 treatments in triplicates with different amounts of manure,urea,calcium phosphate,and potassium chloride.Rhizosphere soil samples of maize and wheat grown in the experimental field in Shandong Province,China,were collected in September 2003 and May 2004,respectively.Arbuscular mycorrhizal fungal spores were isolated and identified using morphological characters.Mycorrhizal colonization percentage,spore density(SD),species richness(SR),relative abundance(RA),and Shannon-Weiner index(SWI) were determined.Nineteen recognized species of AMF belonging to 5 genera were identified.Long-term fixed fertilization significantly influenced colonization percentage,SR,SD,and species diversity of AMF.The adaptability of AMF to soil fertility was different among species.Species richness and SD of AMF in maize and wheat rhizosphere soils were the highest in the nonferti-lization treatment(control) and lowest in the high manure + high nitrogen treatment(M2N2).The SWI decreased as the fertilization level increased except in the low manure treatment(M1) on maize.Compared with the other treatments,Treatment M2N2 significantly reduced SD of Glomus,and the high manure + low nitrogen treatment(M2N1) significantly retarded sporulation of Scutellospora.Manure treatments stimulated sporulation of Glomus mosseae.Spore density of G.mosseae was higher in the high nitrogen + phosphorus + potassium treatment(N2PK) than in the high nitrogen + phosphorus treatment(N2P) and the high nitrogen + potassium treatment(N2K).The SD of S.pellucida was higher in Treatment N2K than Treatments N2PK and N2P.In conclusion,long-term fixed fertilization,especially with high levels of manure and N,decreased SR,SD,and colonization and changed the species composition of AMF.

Production of low cost <i>Bacillus thuringiensis</i>based-biopesticide for management of chickpea pod-borer <i>Helicoverpa armigera</i>(Huebn) in Pakistan

The biopesticide was prepared from locally available low cost ingredients: dried beef blood, molasses and mineral salts (ZnCl2, MgCl2, MnCl2, CaCl2CaCl2, and FeCl3) which were used as medium for the laboratory scale production of Bacillus thuringiensis (B.t.) bio-pesticide by shake flask technique. Indigenous B.t. isolate PA-Sb- 46.3 which produced two crystals—bi-pyramidal and cuboidal was found 73.6 times toxic against H. armigera than reference strain Bacillus thuringiensis var. kurstaki (HD-I-S-1980) used. Medium was fermented for 72 hours at 30℃ ± 2℃ and 160 rpm. 72 h fermented medium showed 95% - 99% sprulation, with spore yield of 3.97 × 109 spores/ml, and LC50 value to 1st instar larvae of H. armigera was 0.53 μg/ml diet. Preservatives and diluents used in the biopesticide were found to be effective to store at room temperature over a period of 30 months. These observations suggested that the biopesticide produced was effective and highly economical for the industrial scale production to manage H. armigera in Pakistan.

Impact of Climate Change on Diseases of Crops and Their Management-A Review

Change in global climate is primarily due to rising concentrations of greenhouse gases in the atmosphere that is mostly caused by human activities.The important factors affecting the occurrence and spread of the plant diseases are temperature,moisture,light,and CO_(2) concentration.These factors cause physiological changes in plants that result in increase in intensity of crop diseases.Climate change causes a significant impact on germination,reproduction,sporulation and spore dispersal of pathogens.Climate change affects all life stages of the pathogen as well as its host to cause impact on host-pathogen interaction which facilitates the emergence of new races of the pathogen ultimately breakdowns the host resistance.It also affects the microbial community in the soil which is beneficial to the plants in various aspects.The minor diseases become major ones due to alteration in climatic parameters thus posing a threat to the food security.

Cultivation Practice of Pleurotus Fossulatus on Rice Straw

Pleurotusfossulatus, a member of oyster mushrooms, was successfully cultivated on rice straw. At least two flushes of fruit body developed at 22 ℃. The first flush of fruit body initiation took place on the 21 st day and second one appeared on 38th day. The biological efficiencies （BE） were found to be 30.8% and 14.6% for first and second flush, respectively. The differentiation pattern of pileus and gills of this mushroom was found to be different from that of other reported species of oyster mushroom. The mushroom （P. fossulatus） took about 20 days for spore formation from primordia initiation and this time span was noted to be highest among all the published reports about any other Pleurotus spp. The BE were 25.9% and 12.5% in first and second flush, respectively when the mushroom was harvested on 18th day （after its initiation of primordia）, i.e., before gill formation. However, the BE was found to be 14-16% lesser in comparison to that harvested after sporulation.

Fight or flee,a vital choice for Clostridioides difficile

Clostridioides difficile is a leading cause of healthcare-associated infections,causing billions of economic losses every year.Its symptoms range from mild diarrhea to life-threatening damage to the colon.Transmission and recurrence of c.difficile infection(CDl)are mediated by the metabolically dormant spores,while the virulence of C.difficile is mainly due to the two large clostridial toxins,TcdA and TcdB.Producing toxins or forming spores are two different strategies for C.difficile to cope with harsh environmental conditions.It is of great significance to understand the molecular mechanisms for C.difficile to skew to either of the cellular processes.Here,we summarize the current understanding of the regulation and connections between toxin production and sporulation in C.difficile and further discuss the potential solutions for yet-to-be-answered questions.

Clinical and microbiological characterization of Clostridium difficile infection in a tertiary care hospital in Shanghai, China

Background Over the last decade,Clostridium difficile infection （CDI） has emerged as a significant nosocomial infection,yet little has been reported from China.This study aimed to characterize the clinical and microbiological features of CDI from a hospital in Shanghai.Methods Patients with CDI seen between December 2010 and March 2013 were included in this study,of which clinical data were retrospectively collected.The microbiological features of corresponding isolates were analyzed including genotype by multi-locus sequence typing （MLST）,antimicrobial susceptibility,toxin production,sporulation capacity,biofilm formation,and motility.Results Ninety-four cases of CDI were included during this study period,12 of whom were severe cases.By reviewing the clinical data,all patients were treated empirically with proton pump inhibitor or antibiotics or both,and they were distributed widely across various wards,most frequently to the digestive ward （28/94,29.79％）.Comparing the severe with mild cases,no significant differences were found in the basic epidemiological data or the microbiological features.Among the 94 isolates,31 were toxin A-negative toxin B-positive all genotyped as ST37.They generated fewer toxins and spores,as well as similar amounts of biofilm and motility percentages,but exhibited highest drug resistance to cephalosporins,quinolones,macrolide-lincosamide and streptogramin （MLSB）,and tetracycline.Conclusions No specific clinical genotype or microbiological features were found in severe cases; antimicrobial resistance could be the primary reason for epidemic strains leading to the dissemination and persistence of CDI.

AoATG5 plays pleiotropic roles in vegetative growth,cell nucleus development,conidiation,and virulence in the nematode-trapping fungus Arthrobotrys oligospora

Autophagy is an evolutionarily conserved process in eukaryotes,which is regulated by autophagy-related genes(ATGs).Arthrobotrys oligospora is a representative species of nematode-trapping(NT)fungi that can produce special traps for nematode predation.To elucidate the biological roles of autophagy in NT fungi,we characterized an orthologous Atg protein,AoAtg5,in A.oligospora.We found that AoATG5 deletion causes a significant reduction in vegetative growth and conidiation,and that the transcript levels of several sporulation-related genes were significantly downregulated during sporulation stage.In addition,the cell nuclei were significantly reduced in theΔAoATG5 mutant,and the transcripts of several genes involved in DNA bio-synthesis,repair,and ligation were significantly upregulated.InΔAoATG5 mutants,the autophagic process was significantly impaired,and trap formation and nematocidal activity were significantly decreased.Comparative transcriptome analysis results showed that AoAtg5 is involved in the regulation of multiple cellular processes,such as autophagy,nitrogen metabolism,DNA biosynthesis and repair,and vesicular transport.In summary,our results suggest that AoAtg5 is essential for autophagy and significantly contributes to vegetative growth,cell nucleus development,sporulation,trap formation,and pathogenicity in A.oligospora,thus providing a basis for future studies focusing on related mechanisms of autophagy in NT fungi.

Diversity and delimitation of Rhizopus microsporus

Rhizopus microsporus has been used for centuries in the production of oriental fermented foods,but the species is also known as a toxin producer and from severe human infections.To study the diversity and species delimitation of Rhizopus microsporus,48 isolates from the reference collection of the CBS-KNAW Fungal Biodiversity Centre,comprising nine environmental,ten clinical,and 23 foodborne strains,in addition to six strains from unknown sources and representing all existing varieties of the species,were examined.Sequence diversity was based on the internal transcribed spacer(ITS),and on a part of the actin(ACT)and translation elongation factor 1-α(TEF)genes.Differences in physiological properties were assessed including temperature relationships.Spore morphology was studied,mating type tests were performed,and MALDI-ToF profiles were generated.Clinical and food-associated strains as well as members of different varieties mated successfully and consequently they belong to a single biological species.Molecular differences did not match with any other parameter investigated.Based on these results the varieties of Rhizopus microsporus are reduced to synonyms.