Evaluation of Streptomyces for Biocontrol of Bipolaris sorokiniana and Sclerotinia homoeocarpa on the Phylloplane of Poa pratensis1

Several species of Streptomyces were evaluated for their ability to control Sclerotinia homoeocarpa (dollar spot) and Bipolaris sorokiniana (leaf spot) on the phylloplane of Poa pratensis (Kentucky bluegrass). Species evaluated included S. diastaticus (S32), S. galbus (S35), and S. hygroscopicus (isolates S13, S28). All evaluations were conducted on the upper epidermis of intact attached leaves of P. pratensis, and antagonism was measured as the ability of Streptomyces isolates to prevent chlorophyll loss from leaves inoculated with B. sorokiniana or S. homoeocarpa during pathogenesis. Only S. hygroscopicus (S13) effectively prevented infection and subsequent chlorophyll loss from leaves inoculated with B. sorokiniana or S. homoeocarpa. Isolate S28 of S. hygroscopicus showed erratic antagonism of both pathogens, depending upon how the isolate was prepared for use. Streptomyces diastaticus and S. galbus were antagonistic to S. homoeocarpa only in whole culture form.     

Influence of Seed‐borne Cochliobolus sativus (Anamorph Bipolaris sorokiniana) on Crown Rot and Root Rot of Barley and Wheat

The effect of seed‐borne pathogens of wheat and barley on crown and root rot diseases of seven barley cultivars (Jimah‐6, Jimah‐51, Jimah‐54, Jimah‐58, Omani, Beecher and Duraqi) and three wheat cultivars (Cooley, Maissani and Shawarir) was investigated. Bipolaris sorokiniana and Alternaria alternata were detected in seeds of at least eight cultivars, but Fusarium species in seeds of only two barley cultivars (Jimah‐54 and Jimah‐58). Crown rot and root rot symptoms developed on barley and wheat cultivars following germination of infected seeds in sterilized growing media. Bipolaris sorokiniana was the only pathogen consistently isolated from crowns and roots of the emerging seedlings. In addition, crown rot and root rot diseases of non‐inoculated barley cultivars correlated significantly with B. sorokiniana inoculum in seeds (P = 0.0019), but not with Fusarium or Alternaria (P > 0.05). These results indicate the role of seed‐borne inoculum of B. sorokiniana in development of crown rot and root rot diseases. Pathogenicity tests of B. sorokiniana isolates confirmed its role in inducing crown rot and root rot, with two wheat cultivars being more resistant to crown and root rots than most barley cultivars (P < 0.05). Barley cultivars also exhibited significant differences in resistance to crown rot (P < 0.05). In addition, black point disease symptoms were observed on seeds of three barley cultivars and were found to significantly affect seed germination and growth of some of these cultivars. This study confirms the role of seed‐borne inoculum of B. sorokiniana in crown and root rots of wheat and barley and is the first report in Oman of the association of B. sorokiniana with black point disease of barley.     

Induced accumulation of tyramine,serotonin, and related amines in response to Bipolaris sorokiniana infection in barley

The inducible metabolites were analyzed in barley leaves inoculated with Bipolaris sorokiniana, the causal agent of spot blotch of barley. HPLC analysis revealed that B. sorokiniana-infected leaves accumulated 4 hydrophilic compounds. They were purified by ODS column chromatography and preparative HPLC. Spectroscopic analyses revealed that they were tyramine (1), 3-(2-aminoethyl)-3-hydroxyindolin-2-one (2), serotonin (3), and 5,5′-dihydroxy-2,4′-bitryptamine (4). Among these, 2 and 4 have not been reported as natural products. They showed antifungal activity in an assay of inhibition of B. sorokiniana conidia germination, suggesting that they play a role in the chemical defense of barley as phytoalexins. The accumulation of 1–4 was examined also in the leaves of rice and foxtail millet. Rice leaves accumulated 2, 3, and 4, whereas foxtail millet leaves accumulated 3 and 4 in response to pathogen attack, suggesting the generality of accumulation of 3 and 4 in the Poaceae species.     

The discovery of the virulence gene ToxA in the wheat and barley pathogen Bipolaris sorokiniana

Bipolaris sorokiniana is the causal agent of multiple diseases on wheat and barley and is the primary constraint to cereal production throughout South Asia. Despite its significance, the molecular basis of disease is poorly understood. To address this, the genomes of three Australian isolates of B. sorokiniana were sequenced and screened for known pathogenicity genes. Sequence analysis revealed that the isolate BRIP10943 harboured the ToxA gene, which has been associated previously with disease in the wheat pathogens Parastagonospora nodorum and Pyrenophora tritici‐repentis. Analysis of the regions flanking ToxA within B. sorokiniana revealed that it was embedded within a 12‐kb genomic element nearly identical to the corresponding regions in P. nodorum and P. tritici‐repentis. A screen of 35 Australian B. sorokiniana isolates confirmed that ToxA was present in 12 isolates. Sequencing of the ToxA genes within these isolates revealed two haplotypes, which differed by a single non‐synonymous nucleotide substitution. Pathogenicity assays showed that a B. sorokiniana isolate harbouring ToxA was more virulent on wheat lines that contained the sensitivity gene when compared with a non‐ToxA isolate. This work demonstrates that proteins that confer host‐specific virulence can be horizontally acquired across multiple species. This acquisition can dramatically increase the virulence of pathogenic strains on susceptible cultivars, which, in an agricultural setting, can have devastating economic and social impacts.     

Transcriptome-based analysis of resistance mechanisms to Bipolaris sorokiniana,a common wheat root-rot disease

• In common root and crown rot (CRR), Bipolaris sorokiniana (Sace.) is one of the important in wheat, causing considerable yield losses globally. Sources of resistance can provide a feasible and effective method of control for plant disease management. However, knowledge on mechanisms of resistance is scarce.
• We screened 33 wheat genotypes against B. sorokiniana under greenhouse and field conditions. In addition, real-time quantitative PCR (qPCR) analysis using ten novel candidate gene markers, Cre3, EDS1, LTP5, PGIP, PR-1, PIEP1, TLP, UGT, Stb6 and PFT, was conducted on leaves and roots, along with changes in activity of antioxidant enzymes, peroxidase, catalase, β-1,3-glucanase, and phenolic content for their involvement in disease impact mechanisms.
• Lowest disease severity was in ‘Alvand’, followed by ‘Baharan’ and ‘Bam’ as resistant genotypes. Quantitative gene expression showed that, although the candidate defence genes were upregulated 1.24- to 3.5-fold in wheat roots and leaves inoculated with B. sorokiniana, they were highly regulated in resistant varieties ‘Alvand’, ‘Mehregan’ and ‘Bam’. Cre3, a resistance gene to cereal cyst nematode Heterodera filipjevi, was regulated in cultivars resistant to B. sorokiniana. Similar results were obtained for Stb6, a gene resistant to Septoria tritici blotch, EDS1 resistant to powdery mildew, Blumeria graminis, and the genes PR-1 and UGT resistant to leaf rust, Puccinia triticina. Antioxidant enzyme activity also showed the highest increases in resistant genotypes.
• In conclusion, the T. aestivum–B. sorokiniana interaction in resistant wheat cultivars uses defence-related genes and enzymes that protect wheat towards sustainable development. Further such studies will shed light on simultaneous resistance to other diseases in wheat cultivars.

     

Vegetative growth and sporulation ofBipolaris sorokiniana on sequentially older infected leaves ofPoa pratensis exposed to postemergence herbicides

Poa pratensis was exposed to the postemergence herbicides 2,4-D, MCPP, and dicamba at concentrations of 10^–6 M applied to the soil surface. Sequentially older leaf blades were inoculated withBipolaris sorokiniana and detached to determine the effect of the herbicides on saprophytic hyphal growth and sporulation on the tissue of each older leaf. 2,4-D and dicamba stimulated hyphal growth in vitro from leaf tissue of all ages; MCPP had no effect on hyphal growth. All herbicides increased sporulation byB. sorokiniana on leaf tissue of all ages. Both hyphal growth and sporulation increased progressively on tissue from sequentially older leaves ofP. pratensis exposed to dicamba.Journal Paper No. J-14985 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa, USA, Project No. 2616.     

URP‐based DNA Fingerprinting of Bipolaris sorokiniana Isolates Causing Spot Blotch of Wheat

Spot blotch, caused by the pathogen Bipolaris sorokiniana is an important disease of wheat and is responsible for large economic losses world wide. In this study, molecular variability in B. sorokiniana isolates collected from different regions of India was investigated using URP‐PCR technique. All the 40 isolates used in the study were pathogenic when tested on susceptible host, Agra local, although they varied in pathogenicity. Isolate BS‐49 was least virulent showing 4.5 infection index while BS‐75 was the most virulent with 63.4 infection index. The universal rice primers (URPs’) are primers which have been derived from DNA repeat sequences in the rice genome. Out of the 12 URP markers used in the study, 10 markers were effective in producing polymorphic fingerprint patterns from DNA of B. sorokiniana isolates. The analysis of entire fingerprint profile using unweighted pair group method with arithmetic averages (UPGMA) differentiated B. sorokiniana isolates obtained from different geographic regions. One isolate BS‐53 from northern hill zone was different from rest of the isolates showing less than 50% similarity. Broadly, three major clusters were obtained using UPGMA method. One cluster consisted of isolates from North western plain zone; second cluster having isolates from North eastern plain zone and third cluster consisted of isolates from Peninsular zone showing more than 75% genetic similarity among them. One of the markers, URP‐2F (5′GTGTGCGATCAGTTGCTGGG3′) amplified three monomorphic bands of 0.60, 0.80 and 0.90 kb size which could be used as specific markers for identification of B. sorokiniana. Further, based on URP‐PCR analysis, the grouping of the isolates according to the geographic origin was possible. This analysis also provided important information on the degree of genetic variability and relationship between the isolates of B. sorokiniana.     

Restriction analysis of the amplified ribosomal DNA spacers ITS1 and ITS2 of <Emphasis Type="Italic">Bipolaris sorokiniana</Emphasis> isolates

The purpose of this study was to examine the genotypic variability of Bipolaris sorokiniana by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis using rDNA. Fifty B. sorokiniana isolates from Brazil and other countries, one Bipolaris oryzae and six Drechslera teres isolates were used. The intragenic spacer regions (ITS1 and ITS2) were the regions used for characterization of isolates. The amplification products for both ITS regions, showed two DNA fragments for all isolates. Two B. sorokiniana isolates presented an intraspecific variability showing a third fragment for the ITS1 region. The dendrograms generated with PCR-RFLP data showed intra- and inter-specific groups. The dendrograms showed that most of Brazilian isolates clustered together forming groups between them, and this behavior was repeated with most isolates from other countries. The dendrograms did not enable the separation of B. sorokiniana isolates by their geographic origin or host type. These results suggest the occurrence of gene flow between different populations of the fungus isolated in geographically distant regions and lends cogency to the occurrence of gene flow between species.     

Vegetative Incompatibility Among Monoconidial Isolates of <Emphasis Type="Italic">Bipolaris sorokiniana</Emphasis>

Bipolaris sorokiniana is a phytopathogenic fungus that causes diseases of cereal crops, such as leaf-spot disease, common root rot, and black point of grain. Because of its great morphological, physiological, and genetic variability, this fungus is difficult to control. The aim of this investigation was to study the variability of isolates of B. sorokiniana by means of vegetative incompatibility. Thirty-five isolates of B. sorokiniana from different geographical regions in Brazil and other countries were used. The vegetative incompatibility between the isolates and the influences of different culture media on these reactions were evaluated. The total protein profile of the isolates was analyzed when the isolates were cultured separately, and in cultures of compatibility and incompatibility reactions. Eighteen of 31 confrontations showed vegetative incompatibility. The results obtained with different culture media for the vegetative compatibility/incompatibility genotypes suggested that the type of substratum influences these reactions. No differences in protein profiles among the isolates were observed. This result suggests that there is no induction of expression of different proteins in vegetative incompatibility reactions.     

The influence of Bipolaris sorokiniana and Drechslera dictyoides metabolites on cell membrane permeability of Festuca pratensis callus

The aim of this study was the evaluation of membrane permeability of callus cells of several Polish meadow fescue cultivars, which were treated with toxins of two leaf spot pathogens Bipolaris sorokiniana and Drechslera dictyoides. Fungus metabolites were obtained by the method described by Lepoivre et al. (1986). Calli of cultivars ‘Skrzeszowicka’, ‘Skawa’, ‘Westa’, POB 282, POB 383, KOA 186 have been selected on medium with metabolites for two weeks. Next the conductivity test of electrolyte leakage and of total ion contents in the examined tissue was done. On the base of this data the membrane permeability coefficients for each cultivar were calculated. Toxins of B. sorokiniana damaged the cell membranes more strongly than metabolites of D. dictyoides. The significant differences of several objects sensitivity to the influence of B. sorokiniana metabolites were stated. These differences were not observed in the case of the influence of D. dictyoides metabolites on the examined tissue.     

Phenylalanine Ammonia-lyase Activity in Barley After Infection with Bipolaris sorokiniana or Treatment with its Purified Xylanase

Phenylalanine ammonia-lyase (PAL) activity was determined from leaves and roots of two barley (Hordeum vulgare L.) cultivars after infection with a necrotrophic pathogen, Bipolaris sorokiniana (Sacc.) Shoem., and treatment with its purified xylanase. PAL activity increased in leaves of both cultivars 16 h after fungal inoculation but two phases, with activity peaks at 24–32 h and 40 h, were recorded only for the more resistant cultivar, Agneta. Attempts to use a PAL inhibitor, χ-amin, ooxyacetic acid, to increase susceptibility to B. sorokiniana in barley leaves were unsuccessful. Treatments of leaves with purified xylanase resulted in more rapid (4–12 h after injection), although reduced, induction of PAL compared with fungal injection. The higher the concentration of xylanase applied the earlier the activity peaks were detected. Fungal inoculation only slightly increased PAL activity in barley roots while xylanase treatment had no effect. The basal level of PAL was however much higher in roots than in leaves. In wheat, Triticum aestivum L. resistant to B. sorokiniana, the time-course of PAL induction after fungal infection and xylanase treatment resembled that for cv. Agneta, while in oats, Avena sativa L. (non-host) PAL activity did not change after the treatments. The results suggest that the second phase of PAL induction, associated only with responses of barley cv. Agneta and wheat, is linked with their resistance to B. sorokiniana infection. The possible role of xylanase as an elicitor of PAL is discussed.     

A novel effector,CsSp1, from Bipolaris sorokiniana,is essential for colonization in wheat and is also involved in triggering host immunity

The hemibiotrophic pathogen Bipolaris sorokiniana causes root rot, leaf blotching, and black embryos in wheat and barley worldwide, resulting in significant yield and quality reductions. However, the mechanism underlying the host–pathogen interactions between B. sorokiniana and wheat or barley remains unknown. The B. sorokiniana genome encodes a large number of uncharacterized putative effector proteins. In this study, we identified a putative secreted protein, CsSp1, with a classic N-terminal signal peptide, that is induced during early infection. A split-marker approach was used to knock out CsSP1 in the Lankao 9-3 strain. Compared with the wild type, the deletion mutant ∆Cssp1 displayed less radial growth on potato dextrose agar plates and produced fewer spores, and complementary transformation completely restored the phenotype of the deletion mutant to that of the wild type. The pathogenicity of the deletion mutant in wheat was attenuated even though appressoria still penetrated the host. Additionally, the infectious hyphae in the deletion mutant became swollen and exhibited reduced growth in plant cells. The signal peptide of CsSp1 was functionally verified through a yeast YTK12 secretion system. Transient expression of CsSp1 in Nicotiana benthamiana inhibited lesion formation caused by Phytophthora capsici. Moreover, CsSp1 localized in the nucleus and cytoplasm of plant cells. In B. sorokiniana-infected wheat leaves, the salicylic acid-regulated genes TaPAL, TaPR1, and TaPR2 were down-regulated in the ∆Cssp1 strain compared with the wild-type strain under the same conditions. Therefore, CsSp1 is a virulence effector and is involved in triggering host immunity.     

Esterase electrophoretic analysis to distinguish isolates between Bipolaris spp. and Drechslera tritici-repentis from wheat

Diseases caused in wheat by Bipolaris sorokiniana and Drechslera tritici-repentis have led to considerable yield and production losses. In wheat seeds another isolate has recently been identified, resembling Bipolaris bicolor. The objective of the present trial was to differentiate and identify isolates of these fungi based on electrophoretic analyses and morphology. Esterase electrophoresis enabled the differentiation between Drechslera sp. and Bipolaris sp. isolates. In relation to morphology, conidia from D. tritici-repentis isolates were significantly longer than the isolates of B. sorokiniana. Bipolaris bicolor isolates, on the other hand, presented wider conidia than those of D. tritici-repentis and B. sorokiniana.     

Preliminary studies on biological control of the blackpoint complex of wheat in Argentina

Blackpoint is a brownish or black discoloration of wheat kernels and biological control is a complementary strategy to manage the disease. This work evaluated the effect of five strains of Trichoderma harzianum and one strain of T. koningii on the growth of Bipolaris sorokiniana and Alternaria alternata and compared the results of screening tests under controlled conditions and field evaluations on bread and durum wheat ears. Disease incidence, infection percentage and seedling emergence percentage determined in a greenhouse assay were evaluated. Dual cultures showed Trichoderma spp. inhibited significantly the mycelial growth of B. sorokiniana between 36 and 71% and of A. alternata between 41 and 61%. Microscopic examination of B. sorokiniana and A. alternata showed plasmolysis and vacuolization of hyphae of the pathogens in the presence of the antagonists tested. With pre-inoculation of wheat ears at anthesis under field conditions, disease incidence, infection percentage by blotter tests and seedling emergence in the greenhouse did not show significant differences between controls and treatments with Trichoderma spp. This revised version was published online in August 2006 with corrections to the Cover Date.     

乙酸钠调控小球藻生长及代谢产物

【背景】小球藻是一种单细胞绿藻,在不同培养条件下可积累高附加值的代谢产物,这些产物可用于生产生物燃料、食品、保健品、药品等。然而这些代谢产物在藻细胞中的生产率较低且很难通过经济可行的方法将其分离,这使其工业化规模生产受到限制。【目的】研究乙酸钠对小球藻生物量的影响,并分析其对小球藻代谢产物的调控作用。【方法】通过在小球藻培养液中添加不同浓度的乙酸钠(1.0、2.0、3.0、4.0、5.0 g/L),研究其调控小球藻生长和代谢的作用机理。【结果】在添加3.0 g/L乙酸钠的培养液中,小球藻的生物量是对照组的5.2倍,尽管藻细胞中蛋白质含量无明显变化,但油脂和类胡萝卜素含量是对照组的2.4倍和1.2倍,多糖和叶绿素a含量却仅为对照组的54.6%和54.4%。【结论】乙酸钠不仅会影响藻细胞的生长,还会调控其代谢过程,这为深入探索乙酸钠在调控小球藻生长及代谢过程的作用机制提供了理论基础和技术资料。     

Biosorption of Cu(II) ions from synthetic and actual wastewater using three algal species

The use of three freshwater microalgal cultures—Chlorella sorokiniana, Anabaena laxa, and Hapalosiphon welwitschii—for sorption of copper(II) from synthetic Cu(II) solutions and Marinduque, Philippines, wastewater was studied. The optimum amount of biomass for the three species was 0.025 g dry weight. The optimum contact time for both C. sorokiniana and A. laxa was 1 h, whereas that of H. welwitschii was 30 min. All three species exhibited maximum Cu(II) sorption at pH 4.0–6.0. The Langmuir adsorption isotherm was the best fit model for the three species. The three cultures were found to be effective biosorbents when used in synthetic wastewaters of low concentration (10–30 ppm). Maximum Cu(II) reductions obtained were 88.2, 88.6, and 91.7% for the C. sorokiniana, A. laxa, and H. welwitschii cultures, respectively. C. sorokiniana, A. laxa, and H. welwitschii removed 5.70, 11.16, and 7.15% of Cu(II), respectively, when applied to wastewater taken from Consolidated Mines Inc. (CMI) containing around 150 ppm Cu(II). C. sorokiniana and A. laxa, in combination, exhibited 14.05% Cu(II) removal from CMI wastewater. Desorption with 0.11 M HCl effected 73.20, 64.54, and 70.85% removal of Cu(II) from the surfaces of C. sorokiniana, A. laxa, and H. welwitschii, respectively. SEM-EDS spectra of the three species confirmed the presence of Cu(II) on their surfaces. Presented at the 6th Meeting of the Asia Pacific Society of Applied Phycology, Manila, Philippines.     

Single nucleotide polymorphism-mismatch primer development for rapid molecular identification of selected microalgal species

Microalgae have been a great source for food, cosmetic, pharmacological, and biofuel production. The adoption of effective diagnostic assays for monitoring all stages of algal cultivation has become essential. In addition to microscopy identification, molecular assays can aid greatly in the identification and monitoring of algal species of interest. In this study the 18S ribosomal RNA (rRNA) sequences of 12 microalgal species and/or strains were used to design algal identification primers. Sequence alignment revealed five highly variable regions and multiple unique single nucleotide polymorphisms (SNPs). To design target algae specific primers, a SNP identified as unique to each microalgal species was incorporated into the 3’-terminus of forward and reverse primer pairs, respectively. To further enhance primer specificity, transverse mutation was introduced into each primer at the third base upstream of the respective SNP. The SNP-mismatch primer pairs yield size-specific amplicons, enabling the rapid molecular detection of 12 microalgae by circumventing cloning and sequencing. To verify the primer specificity, two SNP-mismatch primer pairs designed for Chlorella sorokiniana DOE1412 and wildtype species of Scenedesmus were tested in the outdoor reactor run inoculated with C. sorokiniana DOE1412. The primer pairs were able to identify C. sorokiniana DOE1412 as well as the environmental invader Scenedesmus sp. Furthermore, the “relative concentration” of two microalgae was accessed throughout the entire cultivation run. The use of SNPs primers designed in this study offers a cost-effective, easy to use alternative for routine monitoring of microalgal cultures in laboratories, in scale-ups, and in cultivation reactors, independent of the production platform.

     

Morphology,Physiology, and Virulence of <Emphasis Type="Italic">Bipolaris sorokiniana</Emphasis> Isolates

Bipolaris sorokiniana is a phytopathogenic fungus that causes diseases in cereal crops. The high morphological, physiological, and genetic variability makes the control of this fungus a difficult task. The aim of this work was to study the virulence, morphological, and physiological variability of B. sorokiniana isolates. For this, 35 B. sorokiniana isolates from different geographic regions in Brazil and other countries were used. The isolates were evaluated for their morphological variability, considering mycelium color, sector formation, and growth rate. Based on these morphological characteristics, the isolates were grouped in five different morphological groups. Extracellular enzymes activity in solid medium, virulence in wheat seeds and seedlings, and analysis of total proteins by SDS-PAGE were evaluated for all isolates. Variations among the isolates were found for enzymatic activity, and esterase was the enzyme that showed the highest activity indices. The results obtained from infection of seeds and seedlings showed that isolates from the same geographical region and morphological group had different degrees of virulence. The total protein profile shown by the isolates varied in the number of bands and intensity, where some of them may be used to characterize the specie.