Evidence for a negative membrane potential and for movement of C1- against its electrochemical gradient in the ascomycete Neocosmospora vasinfecta.

The iodides of three lipid-soluble cations (dibenzyldimethylammonium; tribenzylmethylammonium, TBMA+; ethyldimethylbenzylammonium) were synthesized by the reaction of 14C-labeled methyl or 14C-labeled ethyl iodide with the appropriate secondary of tertiary amine and used in an attempt to measure the transmembrane electrical potential difference in Neocosmospora. Only mycelium containing high levels of Na+ accumulated measureable amounts of these cations and only above pH 6. Uptake was reduced in the presence of exogenous K+, Na+, Mg2+, or tris(hydroxymethyl)aminomethane. The velocity of TBMA+ uptake was proportional to its concentration between 46 and 427 muM. Neither the rate nor the extent of TBMB+ uptake was greatly affected by the presence of a fivefold excess of either dibenzyldimethylammonium or ethyldimethylbenzylammonium, even though these cations were themselves accumulated. The uncoupler m-chlorophenylhydrazone induced loss of previously accumulated TBMA+ from the mycelium. Anaerobiosis and cold (5 degrees C) temperature both inhibited TBMA+ uptake but did not induce the loss of previously accumulated TBMA+. The uptake of lipophilic cations by Na+-rich mycelium indicated a minimum transmembrane electrical potential of -60 to -70 mV (inside negative). Net uptake of these cations appeared to be strongly influenced by the availability of endogenous exchangeable cations and by the presence of other exogenous cations, as well as by the membrane potential. Despite these limitations, transport of C1- by Na+-rich mycelium appeared to take place against the electrochemical gradient for C1-.     

In vitro Antagonistic Activity of Endophytic Fungi Isolated from Shirazi Thyme (Zataria multiflora Boiss.) against Monosporascus cannonballus

Endophytic fungi viz., Nigrospora sphaerica (E1 and E6), Subramaniula cristata (E7), and Polycephalomyces sinensis (E8 and E10) were isolated from the medicinal plant, Shirazi thyme (Zataria multiflora). In in vitro tests, these endophytes inhibited the mycelial growth of Monosporascus cannonballus, a plant pathogenic fungus. Morphological abnormalities in the hyphae of M. cannonballus at the edge of the inhibition zone in dual cultures with N. sphaerica were observed. The culture filtrates of these endophytes caused leakage of electrolytes from the mycelium of M. cannonballus. To our knowledge, this is the first report on the isolation and characterization of fungal endophytes from Z. multiflora as well as their antifungal effect on M. cannonballus.Key words: Zataria multiflora, antifungal, endophytic fungi, Monosporascus cannonballus

The term “Endophytes” denotes microorganisms that colonize plants’ internal tissues for part of or throughout their life cycle without producing any apparent adverse effect. The endophytic microorganisms include fungi, bacteria, and actinobacteria (Bacon and White 2000). Among them, fungi are the most common endophytic microorganisms (Staniek et al. 2008). Endophytic fungi are ecologically distinct polyphyletic groups of microorganisms, mostly belonging to the Ascomycota phylum (Jia et al. 2016). Several fungal endophytes have been shown to act as biological control agents for managing soil-borne plant pathogens (Toghueo et al. 2016).Zataria multiflora Boiss. (Synonyms: Zataria bracteata Boiss.; Zataria multiflora var. elatior Boiss), belonging to the Lamiaceae family is a traditional medicinal plant commonly used as a flavor ingredient in different types of foods (Sajed et al. 2013). Several medicinal properties of Z. multiflora, including antiseptic, anesthetic, antispasmodic, antioxidant, antibacterial, and immunomodulatory activities, have been documented (Sajed et al. 2013). However, studies on the endophytic microorganisms inhabiting Z. multiflora are limited (Mohammadi et al. 2016).Monosporascus cannonballus Pollack & Uecker (Ascomycota, Sordariomycetes, Diatrypaceae) is one of the most important phytopathogenic fungi causing root rot and vine decline disease in muskmelon. It causes sudden wilt and collapse of the plant at the fruiting stage, which may result in total yield loss (Martyn and Miller 1996). The fungus also infects pumpkin, cucumber, courgette, and watermelon plants (Mertely et al. 1993). The control of M. cannonballus in melon and other cucurbit crops is difficult because of the pathogen’s soil-borne nature. Earlier reports indicated that arbuscular mycorrhizal fungi (AMF) (Aleandri et al. 2015), hypovirulent isolates of M. cannonballus (Batten et al. 2000), Trichoderma spp. (Zhang et al. 1999), and antagonistic rhizobacteria (Al-Daghari et al. 2020) are effective agents for the reduction of M. cannonballus-induced root rot and vine decline of melon. In addition, it is well established that many endophytic fungi isolated from medicinal plants possess antimicrobial activity against phytopathogenic fungi (Jia et al. 2016). The objective of this study was to investigate the presence of endophytic fungi in Z. multiflora and to study theirs in vitro antagonistic activity against M. cannonballus.Z. multiflora plants (accession number 201100114) were obtained from Oman Botanic Garden, Al-Khoud, Sultanate of Oman. The plants were healthy, showing no apparent symptoms of any disease or pest infestation. A virulent isolate of M. cannonballus (ID14367), obtained from the roots of a melon plant showing root rot and vine decline (Al-Rawahi et al. 2018) was used in this study. The culture was maintained on potato dextrose agar (PDA) medium (Oxoid Ltd., Basingstoke, UK).To isolate endophytic fungi, Z. multiflora plants were washed in tap water to remove adhering soil particles. The leaves were separated, cut into small pieces, and surface-sterilized by washing in 70% (v/v) ethanol for 1 min and then in 1% (v/v) sodium hypochlorite for 1 min. The plant tissues were then washed 3–4 times with sterilized distilled water. The leaf tissue pieces were further cut into small pieces (0.2–0.5 cm in length) using a sterile scalpel and placed on PDA medium. The plates were incubated at 25 ± 2°C for 7–10 days, and pure cultures of the endophytic fungi were obtained (Lu et al. 2012).DNA was extracted from the mycelia for molecular identification of endophytic fungi according to the method described by Liu et al. (2000). PCR amplification of the Internal Transcribed Spacer (ITS) regions of the fungal rDNA was performed using the primers ITS4 (5’-TCCTCCGCTTATTGATATGC-3’) and ITS5 (5’-GGAAGTAAAAGTCGTAACAAGG-3’) as described by Halo et al. (2018). The PCR products of the expected sizes were sequenced at Macrogen, Seoul, Korea. The sequences were subjected to BLAST searches using the National Center for Biotechnology Information (NCBI) database (http://www.ncbi.nlm.nih.gov).A dual culture technique was used to test the in vitro antagonistic effect of the endophytic fungi against M. cannonballus. A mycelial plug (7-mm diameter) was excised from the fungal endophyte colonies and placed on one side of a PDA plate (90-mm diameter) about 1 cm away from the edge. On the same plate, a 7-mm diameter disc of M. cannonballus was placed on the opposite side at 1 cm distance from the edge. The Petri plates inoculated with M. cannonballus alone were used as control. Four Petri plates per treatment were used. The Petri plates were incubated at 25 ± 2°C, and the radial growth of M. cannonballus was measured after 5–7 days of incubation. The mycelial growth inhibition was calculated using the following formula: % inhibition=[1−(T/C)]×100where C – radial growth of M. cannonballus in the control plate and T – radial growth of M. cannonballus in the dual culture plate (Toghueo et al. 2016).To investigate the antagonistic effects of the endophytic fungi on the morphology of M. cannonballus hyphae, the five-mm agar plug samples of M. cannonballus were excised from the colony edges of inhibition zone in the dual culture plate. The samples for scanning electron microscopy were prepared according to the method reported by Goldstein et al. (2003) and observed with a JEOL (Model: JSM-7800F) scanning electron microscope. The culture of M. cannonballus grown in the absence of endophytic fungi served as control.To perform the electrolyte leakage assay, the endophytic fungi were cultured in 200 ml of Czapek Dox broth (static) in 500 ml conical flasks at room temperature (25 ± 2°C) for 14 days, and the culture filtrates were obtained by filtering through Whatman No. 1 filter paper. Five hundred mg of M. cannonballus mycelium were added to 20 ml of culture filtrate in a glass vial. The conductivity of the suspension was measured at 0, 1, and 3 h after incubation by using a conductivity meter (Halo et al. 2018). There were three replicates per treatment and control.Data from the in vitro growth inhibition and the electrolyte leakage assays were statistically analyzed using general linear model ANOVA using Minitab Statistical Software version 17 (Minitab Inc., State College, USA). When ANOVA revealed significant differences between treatments, means were separated using Tukey’s studentized range test at p ≤ 0.05. Arc sine transformation of data on % mycelial growth inhibition was done prior to analysis.A total of five morphologically distinct fungal endophytes were obtained from the leaves of Z. multiflora. Based on the rDNA ITS sequence analysis, these endophytic fungal (Ascomycota, Sordariomycetes) isolates were identified as Nigrospora sphaerica (Amphisphaeriales, Apiosporaceae) (E1 and E6), Subramaniula cristata (Sordariales, Chaetomiaceae) (E7) and Polycephalomyces sinensis (Hypocreales, Ophiocordycipitaceae) (E8 and E10). The sequences were deposited in the GenBank database (http://www.ncbi.nlm.nih.gov/genbank/) under the accession numbers {"type":"entrez-nucleotide","attrs":{"text":"MH028052","term_id":"1509265912","term_text":"MH028052"}}MH028052, {"type":"entrez-nucleotide","attrs":{"text":"MH028054","term_id":"1509265914","term_text":"MH028054"}}MH028054, {"type":"entrez-nucleotide","attrs":{"text":"MH028055","term_id":"1509265915","term_text":"MH028055"}}MH028055, {"type":"entrez-nucleotide","attrs":{"text":"MH028056","term_id":"1509265916","term_text":"MH028056"}}MH028056, and {"type":"entrez-nucleotide","attrs":{"text":"MH028058","term_id":"1509265918","term_text":"MH028058"}}MH028058. P. sinensis is an important medicinal fungus. Numerous pharmacological activities of P. sinensis including immunomodulatory, anti-estrogenicity and antitumor activities have been documented (Wang et al. 2012). N. sphaerica has been reported as an endophyte (Wang et al. 2017) as well as a pathogen in a few plant species (Wright et al. 2008; Liu et al. 2016). However, Z. multiflora plants colonized with these endophytic fungi were healthy and did not show any observable disease symptoms.The in vitro dual culture antagonism assay showed that all the five endophytic fungi inhibited the mycelial growth of M. cannonballus. N. sphaerica E1 was the most effective (81.7%), followed by P. sinensis E8 (80.6%), P. sinensis E10 (75.8%) and N. sphaerica E6 (66.1%). S. cristata E7 was the least effective, which recorded 38.7% inhibition (Table ​(TableI,I, Fig. ​Fig.1).1). Further, scanning electron microscopic observations of the hyphae of M. cannonballus from the dual culture assay plates at the edge of the inhibition zone revealed morphological abnormalities such as disintegration, shrinkage, and loss of turgidity. Scanning electron micrograph of M. cannonballus after co-cultivation with the endophytic fungus N. sphaerica E1 is shown in Fig. ​Fig.2.2. These findings corroborate with those of Hajlaoui et al. (1992) who reported plasmolysis of Sphaerotheca pannosa var. rosae mycelium due to the antagonistic effect of Sporothrix flocculosa. Halo et al. (2018) reported shrinkage of Pythium aphanidermatum hyphae due to the antagonistic activity of Aspergillus terreus. The shrinkage of M. cannonballus hyphae in the present study suggests a possible leakage of cytoplasmic contents (Garg et al. 2010). The loss of the turgidity of M. cannonballus hyphae indicates alterations in the permeability of the cell membrane (Halo et al. 2018). Several reports indicate the production of antimicrobial substances by endophytic fungi (Zhao et al. 2012; Homthong et al. 2016). Kim et al. (2001) demonstrated that phomalactone, a compound produced by N. sphaerica restricted the mycelial growth and germination of sporangium and zoospore of Phytophthora infestans and decreased the incidence of late blight in tomato. Zhao et al. (2012) characterized four secondary antifungal metabolites viz., dechlorogriseofulvin, griseofulvin, mullein, and 8-dihydroramulosin from the liquid cultures of the endophytic fungus Nigrospora sp. isolated from roots of the medicinal plant, Moringa oleifera. Homthong et al. (2016) reported the production of chitinase by Paecilomyces (Polycephalomyces) sp. The inhibitory effect of endophytic fungi on the hyphae of M. cannonballus in this study might be due to the production of antifungal metabolites.Table IPercentage inhibition of mycelial growth of M. cannonballus by endophytic fungi isolated from Zataria multiflora in dual cultures on PDA.

枯草芽孢杆菌TF26抗菌蛋白抑菌活性的初步研究

目的:研究枯草芽孢杆菌TF26抗菌蛋白的抑菌活性和生物稳定性,为菌株及抗菌蛋白的应用提供理论依据.方法:采用硫酸铵盐析方法提取抗菌蛋白,采用菌丝生长速率法检测其对13种植物病原真菌的抑菌活性,采用抑菌圈方法对其生物稳定性进行分析.结果:抗菌蛋白粗提物能够抑制13种植物病原真菌的生长,平皿抑制率为74.3％ ～91.3％,对葵花菌核病菌、番茄和黄瓜枯萎病菌、黄瓜菌核病菌和立枯病菌、水稻恶苗病菌和大豆根腐病菌抑制作用较强.抗菌蛋白在100℃以下,pH＜ 10范围内抑菌活性稳定,对紫外线照射不敏感,室温(20℃)和4℃储存150d抑菌活性稳定.结论:抗菌蛋白具有较强的热、酸碱、紫外和储存稳定性以及广谱的抑菌活性.     

Antagonistic actions of Pythium oligandrum and Trichoderma harzianum against phytopathogenic fungi (Fusarium oxysporum and Pythium ultimum var. ultimum)

Abstract

The possible biological control of damping-off fungi, Fusarium oxysporum and Pythium ultimum by Pythium oligandrum or Trichoderma harzianum was in vitro investigated. Results of comparing the antagonistic activity of P. oligandrum and T. harzianum in dual plates against the tested phytopathogens indicated different degrees of antagonism. After 12 days of incubation colony of the phytopathogenic fungus was completely overgrown by the antagonist, except for the interaction between T. harzianum and F. oxysporum which showed no overgrowth or any hyphal penetration by the antagonist. However, growth and proliferation of F. oxysporum colony was repressed. T. harzianum and P. oligandrum produced chitinase and β-1,3-glucanase when they were grown on liquid culture medium supplemented with chitin or fungal dried mycelium as a sole carbon source, and enzyme production was higher by T. harzianum comparing with P. oligandrum under the same condition. Fungal dried mycelium of F. oxysporum was the most selective carbon source for enzyme production, on the other hand, chitinase production was significant locked when P. ultimum dried mycelium was used as a carbon source. Production of volatile compounds by P. oligandrum or T. harzianum against F. oxysporum and P. ultimum was examined using the inverted plates method. F. oxysporum was inhibited by the antagonist volatile compounds and it is inhibited 100% by increasing the amount of inoculum size. Production of potential biocontrol agents provided with economically features and working under field conditions are recommended.     

Litsea glaucescens Humb., Bonpl. & Kunth var.Glaucescens (Lauraceae): A Mexican bay

Economic Botany - Thirty-one compounds are identified in the essential oil of Mexican bay (Litsea glaucescens var.glaucescens), which is dominated by 22.36± 2.19% 1,8-cineole, 13.03±...     

Isolation ofGlomerella musae [teleomorph ofColletotrichum musae (Berk. & Curt.) Arx.] and segregation analysis of ascospore progeny

The sexual stage ofColletotrichum musae has been obtained under controlled laboratory conditions. Development of the sexual stage required light and suboptimal growth temperature. Meiosis was apparent as judged by the formation of perithecia typical of ascomycetes classified asGlomerella. Perithecia contained as many as 50–80 asci, each of which contained up to eight ascospores. The ascospores were larger than conidia and were easily dissected for segregation analysis of either random spores or tetrads. In order to determine if this meiotic system was homothallic or heterothallic, chlorate-resistant mutants were isolated and crossed with wild type. Chlorate resistance and susceptibility segregated in a 1:1 ratio, indicating that this system was heterothallic. Sexual recombination was observed by analyzing the segregation of several DNA markers identified by amplification of polymorphic fragments of genomic DNA using single oligonucleotide primers andTaq DNA polymerase. Segregation consistent with mendelian inheritance and sexual recombination of genetic markers indicated that it is feasible to construct a genetic map for this species. The complexity of sexual compatibility was analyzed by crossing several ascospore progeny which identified four distinct mating types.     

Antifungal Activity of Crude Extracts of <i>Tectona grandis</i> L.f. against Wood Decay Fungi

Wood is mainly made up of cellulose, hemicelluloses, lignin polymers and other organic and inorganic substances, making it susceptible to deteriorate by various biological agents. Tectona grandis L.f. (Teak) is a timber species with high resistance to biological deterioration, valued for its durability, beauty, and mechanical resistance. The purpose of this work was to evaluate the antifungal activity of crude extracts from teak on various fungi that cause wood deterioration. For this, Teak heartwood was obtained, then fragmented and pulverized until obtaining a flour which was used for compounds extraction using the Soxhlet technique coupled to a rotary evaporator through solvents of increasing polarity (hexane, dichloromethane, tetrahydrofuran, and acetone). The extracts obtained were tested against fungal organisms collected in the field, and the LC₅₀ was determined using teak crude extracts on Artemia salina as a biological model. The results obtained showed that a high flour yield was obtained with hexane (0.951 g), followed by tetrahydrofuran (0.446 g), dichloromethane (0.348 g), and acetone (0.152 g). By using nine fungal organisms that predominantly correspond to the genus Aspergillus, the extractable compounds were tested, inhibiting 25% of mycelial growth with tetrahydrofuran (T. versicolor), and 40.9% with dichloromethane (G. trabeum). Likewise, the biological model of A. salina showed an LC₅₀ of 84.9 μg/mL with hexane, 43.3 μg/mL with dichloromethane, 59.6 μg/mL with tetrahydrofuran, and 54.7 μg/mL with acetone. For this reason, it is concluded that Teak wood contains many extractable compounds in relation to its weight, besides having antimicrobial activity when extracted through polar compounds such as dichloromethane and tetrahydrofuran.     

番茄青枯病拮抗菌的筛选

利用点接法从山东寿光和苍山不同种植年限的蔬菜大棚中分离得到的45株菌株以及实验室保存的19株菌株中,筛选到14株对番茄青枯病病原细菌有拮抗作用的菌株,然后通过牛津杯法复筛得到6株抑菌效果较好的细菌株。通过温室盆栽试验表明拮抗菌X10的防治效果最好,液体菌剂防治效果达到了81.8%,固体菌剂防治效果达到了65.4%,具有良好的应用前景。对菌株X10进行了培养特征、形态特征和生理生化特征测定,鉴定为侧胞短杆芽胞杆菌(Brevi Bacillus laterosporus)。     

云南重楼植物内生真菌的分离及抗菌活性筛选

从云南重楼[Paris polyphylla Smithvar.yunnnanensis(Franch.)Hand.Mazz.]块状茎中分离出166株内生真菌,对其进行形态分类鉴定归于4目,6科,20个属,体现了云南重楼植物内生真菌的生物多样性特征。同时,选择与人类和植物相关的37株病原微生物作为抗菌活性筛选指示菌,进行了云南重楼植物内生真菌抗菌活性的初步研究。结果表明,4株内生真菌对细菌、植物致病真菌、皮肤致病真菌多种病原微生物具有显著抑制生长的作用。     

Euphrasia amurensis, E. hirtella var. ramosa and var. karoiana (Orobanchaceae)

The lectotype for Euphrasia hirtella var. ramosa is selected. This taxon and E. hirtella var. karoiana are compared with E. amurensis and confirmed as synonyms, corroborating Juzepcuk (1955 ).  © 2007 The Linnean Society of London, Botanical Journal of the Linnean Society , 2007, 154 , 215–223.     

Morphological and cytogenetical characterization of Vicia montbretii Fisch. & Mey. (Synonym: Lens montbretii (Fisch. & Mey.) Davis & Plitmann)

Lens montbretii has a diploid chromosome number of In – 12 whereas the remainder of the genus has 2n= 14. In addition there are marked karyotype differences. These, combined with morphological characteristics which distinguish it from other species of Lens , are considered to be an adequate basis for the returning of the taxon to the genus Vicia as V. montbretii Fisch. & Mey.     

Pollination Ecology and Endemic Trends in Pedicularis bracteosa var. atrosanguinea Pennell & Thompson (Scrophulariaceae) in North America

Abstract Pedicularis bracteosa var. atrosanguinea occurs locally in association with P. racemosa or P. groenlandica in the Olympic Mountains in Washington. Other plant species, e.g., Polygonum bistortoides, Lupinus argenteus var. parviflorus and Valeriana sitchensis compete for space and bumblebee pollinators within the study area. Pollinator sharing, resulting from such competition, may increase the frequency of unvisited flowers of P. bracteosa var. atrosanguinea. P. bracteosa var. atrosanguinea , with blood purple nectariferous flowers, is presumed to lack intense reflections of blue spectral components from its corollas in attracting bumblebees compared to those of P. racemosa and P. groenlandica . Six species of bumblebees ( Bombus ) and cuckoo bees ( Psithyrus ) pollinate P. bracteosa var. atrosanguinea . Of these, Bombus mixtus and B. occidentalis occur in higher frequencies and are the major pollinators of P. bracteosa var. atrosanguinea . Queen and larger worker bumblebees pollinate nototribically as they probe for nectar, while smaller worker bumblebees pollinate sternotribically while scraping pollen from anthers deeply hidden in the dome-shaped galea. Corbicular pollen loads of bumblebees collected in the study area contain Pedicularis pollen alone/in combination of Polygonum, Valeriana, Lupinus, Erigeron and Bidens , or exclusively of Polygonum or Valeriana. P. bracteosa var. atrosanguinea does not suffer seriously from deficient pollination but seedlings resulting from pollinated flowers may be subjected to natural selection pressure exerted by colonial plant species for space. P. bracteosa var. atrosanguinea does not propagate asexually but resumes aerial growth seasonally from the self-same underground root stocks. If seedlings are under continuous selection pressure for lack of space, P. bracteosa var. atrosanguinea is presumed to regenerate primarily by perennial root stocks. This behavior may favor endemism in P. bracteosa var. atrosanguinea .