内生真菌感染对宿主羊草抗病性的影响

以感染内生真菌的天然禾草羊草为实验材料,通过体外纯培养条件下的内生真菌、感染内生真菌的离体叶片和在体叶片对3种病原菌的抑菌实验,以探讨内生真菌对宿主植物羊草在抗病性方面的贡献。结果表明:体外纯培养条件下,分离自羊草的内生真菌Epichlobromicola对新月弯孢(Curvularia lunata)、根腐离蠕孢(Bipolaris sorokiniana)和枝孢霉(Cladosporium sp.)这3种病原菌都具有抑制作用,抑菌率分别达56.22%,46.93%和45.15%,且内生真菌培养滤液可以有效抑制这3种病原菌的孢子萌发,平均萌发率分别为30.4%,15.7%和16.4%;宿主植物叶片在离体条件下,内生真菌感染可以有效降低羊草叶片受C.lunata和C.sp.侵染后的病斑数或病斑长度,但对B.sorokiniana不起作用,甚至提高了叶片的病斑数及病斑长度,而离体叶片提取液对不同病原菌均有不同程度的抑制作用;在体条件下,内生真菌均可以通过降低叶片病斑数来增强羊草植株对这3种病原菌的抗性。由此看来,内生真菌E.bromicola对宿主植物羊草在抗病原菌侵染方面有一定的增益作用。     

Aggressiveness analysis of foliar fungi to leaves of wheat and rice crops

Abstract

The investigations were based on two surveys of wheat and one survey of rice. Alternaria alternata, Curvularia lunata, Helminthosporium tetramera and Bipolaris sorokiniana were isolated and identified from foliage and soil of both wheat and rice crops and their aggressiveness was studied by aggressiveness analysis screened out into different aggressiveness classes. The aggressiveness of isolated fungi was studied on wheat varieties (Inqalab-91 and Chakwal-86) and rice varieties (Basmati-385 and IRRI-6) under controlled conditions. In the foliar aggressiveness test of A. alternata, the overall number of aggressive isolates was higher on wheat varieties than rice. Bipolaris sorokiniana isolates showed foliar blight symptoms on wheat but not on rice varieties. In C. lunata, the overall number of aggressive isolates was higher on wheat. In the foliar aggressiveness test of H. tetramera, the number of non-aggressive isolates was almost the same on wheat and rice varieties. In the present study it became clear that A. alternate, B. sorokiniana, C. lunata and H. tetramera are common foliar pathogens in rice and wheat crops and can cause soil-borne and foliar diseases.     

Diversity and antimicrobial activity of endophytic fungi isolated from Nyctanthes arbor-tristis, a well-known medicinal plant of India

Endophytic fungi from Nyctanthes arbor-tristis were isolated and evaluated for their antimicrobial activity. A total of 19 endophytic fungi were isolated from 400 segments of healthy leaf and stem tissues of N. arbor-tristis. Eighteen endophytic fungi were obtained from leaf, while only ten from stem. Alternaria alternata had the highest colonization frequency (15.0%) in leaf, whereas Cladosporium cladosporioides ranked first in stem with a colonization frequency of 12%. The diversity and species richness were found higher in leaf tissues than in stem. The similarity indices between leaf and stem were 0.473 for Jaccard’s and 0.642 for the Sorenson index, respectively. Of 16, 12 (75%) endophytic fungal extracts showed antibacterial activity against either one or more pathogenic bacteria. The endophytic Nigrospora oryzae showed maximum inhibition against Shigella sp. and Pseudomonas aeruginosa. The leaf endophytes Colletotrichum dematium and Chaetomium globosum exhibited a broad range of anibacterial activity and were active against Shigella flexnii, Shigella boydii, Salmonella enteritidis, Salmonella paratyphi, and P. aeruginosa. Nine out of 16 (56.25%) endophytic fungi exhibited antifungal activity to one or more fungal pathogens. Colletotrichum dematium inhibited 55.87% of the radial growth of the phytopathogen Curvularia lunata. The antimicrobial activity of these endophytic microorganisms could be exploited in the biotechnological, medicinal, and agricultural industries.     

Interactive signal transfer between host and pathogen during successful infection of barley leaves by Blumeria graminis and Bipolaris sorokiniana

Using ion-selective microprobes, interactive signalling between barley and Blumeria graminis or Bipolaris sorokiniana has been investigated. The question was raised whether a biotrophically growing fungus manipulates the electrical driving forces (membrane potential, transmembrane pH), required for H⁺ cotransport of energy-rich compounds. Electrodes were positioned in the substomatal cavity of open stomata or on the leaf surface, and pH was measured continuously up to several days during fungal development. We demonstrate that surface and apoplastic fluids are electrically coupled and respond in a similar manner to stimuli. Apoplastic pH, monitored from the moment of inoculation with conidia, reveals several phases: 2–4 h after inoculation of the barley leaf with either fungus, the host displays rapid transient responses after its first contact with the fungal cell wall; apoplastic pH and pCa increases, cytoplasmic pH and pCa decreases. About 1 day after inoculation, the apoplastic pH increases by up to 2 pH units, which is thought to reflect a resistance response against the intruder. Whereas barley leaf cells possess a membrane potential of −152±5 mV, hyphae of B. graminis yield −251±8 mV, indicative of a substantial driving force advantage for the fungus. Although the resting membrane potential of barley remains constant during the first days after inoculation, leaves infected with B. sorokiniana get confronted with an energy problem, indicated by a retarded repolarization following a “light-off” stimulus. Five days after inoculation, apoplastic pH has increased to 5.97±0.47 (n=11) and does no longer respond to “light-off” when measured within lesions. In contrast, it stays at near normal values outside the lesions and responds to “light-off”.It is concluded that biotrophically growing fungi do not manipulate the cotransport driving forces since (i) any change in apoplastic pH would be experienced by both partners; (ii) the resting membrane potential is not changed. It is suggested that measured pH changes reflect defence responses of the host against the fungus rather than fungal action to increase compatibility.     

Antifungal metabolites (monorden, monocillins I, II, III) from Colletotrichum graminicola, a systemic vascular pathogen of maize

Colletotrichum graminicola is a systemic vascular pathogen that causes anthracnose stalk rot and leaf blight of maize. In the course of an effort to explore the potential presence and roles of C. graminicola metabolites in maize, ethyl acetate extracts of solid substrate fermentations of several C. graminicola isolates from Michigan and Illinois were found to be active against Aspergillus flavus and Fusarium verticillioides, both mycotoxin-producing seed-infecting fungal pathogens. Chemical investigations of the extract of one such isolate (NRRL 47511) led to the isolation of known metabolites monorden (also known as radicicol) and monocillins I–III as major components. Monorden and monocillin I displayed in vitro activity against the stalk- and ear-rot pathogen Stenocarpella maydis while only the most abundant metabolite (monorden) showed activity against foliar pathogens Alternaria alternata, Bipolaris zeicola, and Curvularia lunata. Using LC–HRESITOFMS, monorden was detected in steam-sterilized maize stalks and stalk residues inoculated with C. graminicola but not in the necrotic stalk tissues of wound-inoculated plants grown in an environmental chamber. Monorden and monocillin I can bind and inhibit plant Hsp90, a chaperone of R-proteins. It is hypothesized that monorden and monocillins could support the C. graminicola disease cycle by disrupting maize plant defenses and by excluding other fungi from necrotic tissues and crop residues. This is the first report of natural products from C. graminicola, as well as the production of monorden and monocillins by a pathogen of cereals.     

Identification and Pathogenicity of Fungal Pathogens Causing Black Point in Wheat on the North China Plain

Fungi associated with black point were isolated from three highly susceptible wheat genotypes in the North China Plain. The 21 isolates represented 11 fungal genera. The most prevalent genera were Alternaria (isolation frequency of 56.7%), Bipolaris (16.1%), and Fusarium (6.0%). The other eight genera were Curvularia, Aspergillus, Cladosporium, Exserohilum, Epicoccum, Nigrospora, Penicillium, and Ulocladium; their isolation frequencies ranged from 0.8 to 4.8%. The pathogenicity of the isolates was individually assessed in the greenhouse by inoculating wheat plants with spore suspensions. Ten of the 21 isolates caused significantly higher incidences of black point than that the controls. These isolates belonged to eight fungal species (A. alternata, B. sorokiniana, B. crotonis, B. cynodontis, C. spicifera, F. equiseti, E. rostratum, and E. sorghinum) based on morphological traits and phylogenetic analysis. The average incidences of black point in the eight fungal species were 32.4, 54.3, 43.0, 41.9, 37.2, 38.8, 50.1, and 34.1%, respectively. B. sorokiniana and A. alternata were determined to be the most important pathogens in the North China Plain based on fungal prevalence and symptom severity. This study is the first to identify E. rostratum as a major pathogen causing black point in wheat.     

A comparative study of endophytic and epiphytic fungal association with leaf of <Emphasis Type="Italic">Eucalyptus citriodora</Emphasis> Hook., and their antimicrobial activity

Eucalyptus citriodora Hook, is frequently cultivated tree in India for its wood and medicinal usages. The endophytic and epiphytic fungi were estimated from healthy leaves of E. citriodora growing in the premise of Banaras Hindu University, Varanasi, India. A total of 33 fungal species were isolated from leaf segments. Of 33 taxa, 20 were recorded as endophytes, while 22 as epiphytes. Nine, out of 33 species were found to be common in leaf tissues and surfaces (Alternaria alternata, Aspergillus fumigatus, A. terreus, Cladosporium cladosporioides, Drechslera rostrata, Humicola grisea, Nigrospora oryzae, Penicillium cristata, and Pestalotia sp.). Out of 478 fungal isolates, 279 were epiphytic while only 199 were endophytic. Most isolates were anamorphic filamentous fungi which often don’t produce sexual spores. The Sorensen’s index of similarity between endophytes and epiphytes (leaf surface colonizers) was found to be at 0.300. Diversity index of fungal species was higher in endophytes than epiphytes. The frequency of colonization differs greatly in both myco-populations. Cladosporium cladosporioides (26%) was dominant species on leaf surfaces while Botrytis cinerea (10.5%) was dominant in leaf tissues. Out of 16 endophytic isolates evaluated for antagonistic test, 8 (50%) gave the antagonistic activity against variety of fungi representing pathogens to both humans and plants.     

Identification of Fungal Species Associated with Cladode Spot of Prickly Pear and Their Sensitivity to Chitosan

México is the most important producer of prickly pear (Opuntia ficus‐indica) in the world. There are several fungal diseases that can have a negative impact on their yields. In this study, there was a widespread fungal richness on cladodes spot of prickly pears from México. A total of 41 fungi isolates were obtained from cladodes spot; 11 of them were morphologically different. According to the pathogenicity test, seven isolates caused lesions on cladodes. The morphological and molecular identification evidenced the isolation of Colletotrichum gloeosporioides, Alternaria alternata, Fusarium lunatum, Curvularia lunata. All these species caused similar symptoms of circular cladodes spot. However, it is noticeable that some lesions showed perforation and detachment of affected tissues by Fusarium lunatum. To our knowledge, this is the first report of the Fusarium lunatum as phytopathogenic fungus of cladodes of prickly pear. The chitosan inhibited the mycelium growth in the seven isolates of phytopathogenic fungi. Chitosan applications diminished the disease incidence caused by C. gloeosporioies and F. lunatum in 40 and 100%, respectively. Likewise, the lesion severity index in cladodes decreased. There are no previous reports about the application of chitosan on cladodes of prickly pears for the control of phytopathogenic fungi. Therefore, this research could contribute to improve the strategies for the management of diseases in prickly pear.     

Susceptibility of tall fescue to Rhizoctonia zeae infection as affected by endophyte symbiosis

Endophytic fungi belonging to the genus Neotyphodium often form symbiotic associations with grasses. The host plants usually benefit from the association with an endophyte. Presence of the symbiont may increase host resistance to infection by some pathogens. However, the exact mechanism of the lower susceptibility of endophyte‐infected plants to diseases is still unclear. Growth chamber trials were conducted to determine whether (a) tall fescue plants infected with the endophyte Neotyphodium coenophialum (E+) are more resistant to sheath and leaf spot disease caused by Rhizoctonia zeae than endophyte‐free (E?) plants, and (b) R. zeae growth inhibition is associated with endophyte presence. Tall fescue genotypes, each symbiotic with a genetically different native endophyte strain, were inoculated with isolates of R. zeae. The tillers infection by R. zeae, density of endophyte hyphae and content of total phenolic compounds in tillers were studied. Antifungal activity of the N. coenophialum towards R. zeae, Rhizoctonia solani, Bipolaris sorokiniana and Curvularia lunata was also investigated in dual‐culture assays. For Tf3, Tf4, TfA2 and TfA9 tall fescue genotypes, the E+ plants had reduced R. zeae infection. In the Tf9 and Tf8085 genotypes, R. zeae infection was similar for both E+ and E? plants. The strongest effect was observed for the Tf4 endophyte. A strongly positive correlation (r = 0.94) occurred between endophyte hyphal density and disease index across all tall fescue genotypes. Dual‐culture assays showed no inhibitory interaction between the seven endophyte strains and the R. zeae isolates; however, some endophytes inhibited R. solani, B. sorokiniana and C. lunata. Endophyte presence increased the production of phenolic compounds by the host grasses. The level of phenolics also differed significantly depending on the time of analysis after inoculation of plants by R. zeae. The results indicate that N. coenophialum can suppress disease severity caused by R. zeae infection. The mechanism of higher resistance of E+ plants is likely not based on direct inhibition such as antibiosis or competition. Thus, the induction of specific mechanisms in the host plant, for example, production of phenolic compounds, seems to be the main way of providing resistance to the grass by the endophyte.     

Leaf scarring by the weevils Neochetina eichhorniae and N. bruchi enhances infection by the fungus Cercospora piaropi on waterhyacinth, Eichhornia crassipes

Additive or synergistic effects among introduced and native insect and plant pathogen agents are necessary to achieve biological control of waterhyacinth (Eichhornia crassipes), a globally damaging aquatic weed. In field plots, plants were infested with waterhyacinth weevils (Neoechetina bruchi and N. eichhorniae) and leaves were scarred by weevil feeding. Subsequent infection by the fungal pathogen Cercospora piaropi caused necrotic lesions to form on leaves. Necrosis development was 7.5- and 10.5-fold greater in plots augmented with both weevils and C. piaropi and weevils alone, respectively, than in plots receiving only C. piaropi. Twenty-four days after weevil infestation, the percentage of laminar area covered by lesions on third-youngest and oldest live leaves was elevated 2.3–2.5-fold in plots augmented with weevils. Scar density and necrosis coverage on young leaf laminae were positively correlated, even though antipathogenic soluble peroxidases were elevated 3-fold in plots augmented with weevils alone or weevils and C. piaropi. Combined weevil and fungal augmentation decreased shoot densities and leaves per plant. In a no-choice bioassay, weevil feeding on oldest but not young leaves was reduced 44 two weeks after C. piaropi inoculation. Protein content and peroxidase activities were elevated 2–6-fold in oldest leaves three weeks after inoculation. Augmentation with both waterhyacinth weevils and C. piaropi led to the development of an additive biological control impact, mediated by one or more direct interactions between these agents, and not plant quality effects.     

Small-spored Alternaria spp. (section Alternaria) are common pathogens on wild tomato species

The wild relatives of modern tomato crops are native to South America. These plants occur in habitats as different as the Andes and the Atacama Desert and are, to some degree, all susceptible to fungal pathogens of the genus Alternaria. Alternaria is a large genus. On tomatoes, several species cause early blight, leaf spots and other diseases. We collected Alternaria-like infection lesions from the leaves of eight wild tomato species from Chile and Peru. Using molecular barcoding markers, we characterized the pathogens. The infection lesions were caused predominantly by small-spored species of Alternaria of the section Alternaria, like A. alternata, but also by Stemphylium spp., Alternaria spp. from the section Ulocladioides and other related species. Morphological observations and an infection assay confirmed this. Comparative genetic diversity analyses show a larger diversity in this wild system than in studies of cultivated Solanum species. As A. alternata has been reported to be an increasing problem in cultivated tomatoes, investigating the evolutionary potential of this pathogen is not only interesting to scientists studying wild plant pathosystems. It could also inform crop protection and breeding programs to be aware of potential epidemics caused by species still confined to South America.     

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.     

Definition of leaf health in Agrostis palustris at the time of infection and colonisation by Curvularia lunata

The state of leaf health of intact Penneagle creeping bentgrass leaves into which Curvularia lunata was able to ingress was determined by reducing cuticle/wax formation with trichloroacetic acid (TCA) and stressing plants with high air temperatures. Leaf health was estimated throughout the growing period of the plants by extracting chlorophyll a and then regressing the values with respect to time. In this manner, leaves at each nodal position could be classified as either juvenile, mature or senescent. The addition of TCA to plants decreased leaf and plant life. Stressing plants with high air temperatures accelerated leaf senescence as did higher levels of TCA. Inoculation with Drechslera sorokiniana resulted in lesion formation within 2 days. Symptoms commonly attributed to Curvularia blight were present on plants treated with 140 ^M TCA when high-air-temperature stressed and inoculated with C. lunata. Histochemical procedures failed to show the presence of mycelium of C. lunata within the cells of Curvularia blight symptom areas. Results demonstrated that C. lunata had the ability to colonise heat stressed and/or old leaves but did not have the ability to infect and colonise juvenile or mature tissues.     

Pathogenicity and specificity of Neozygites tanajoae and Neozygites floridana (Zygomycetes: Entomophthorales) isolates pathogenic to the cassava green mite

The cassava green mite (CGM), Mononychellus tanajoa, a native of South America was accidentally introduced into Africa where it causes serious crop losses. The possibility of introducing classical biological agents from the native home of CGM into Africa was investigated. Thus, we conducted a series of laboratory assays of the native fungal pathogens, Neozygites tanajoae from Brazil and Neozygites floridana from Colombia and Brazil, and compared them with N. tanajoae isolates from Benin. Infectivity of both fungal species, was assayed against the twospotted spider mite, Tetranychus urticae, and against the red mite, Oligonychus gossypii. Pathogenicity against CGM and host range studies were conducted by transferring adult females of each mite species to leaf discs containing sporulated cadavers with a halo of conidia of each fungal isolate. All isolates caused some degree of infectivity to CGM. None of the isolates of N. floridana and N. tanajoae tested were pathogenic to O. gossypii, and only two isolates infected T. urticae. Most isolates from Brazil were highly virulent and infected only CGM. Sixteen N. tanajoae isolates caused more than 89% mortality and more than 62% of the CGM became mummified. A mummified CGM is characteristically a swollen, brown fungus-killed mite that has great potential to produce conidia. However, high mortality was not always associated with high mummification. The median mummification time ranged from 4.4 to 6.7 days. Five Brazilian isolates caused >75% mummification with a median mummification time <5 days. Isolates that cause high mummification in a short period of time would be more likely to cause epizootics and to establish in the new environment. Therefore, these isolates would be the best candidates for introduction to Africa.     

Selection of mutants resistant to Alternaria blotch from in vitro -cultured apple shoots irradiated with X- and γ-rays

We produced mutants resistant to Alternaria blotch disease in several cultivars of apple (Malus × domestica Borkh.) by irradiation with X- or γ-rays. An efficient in vitro assay method was established using chemically-synthesized AM-toxin I of Alternaria alternata (Fr.) Keissler to screen for mutants resistant to Alternaria blotch disease. The frequency of necrotic lesions was investigated by applying various concentrations of AM-toxin I to leaf discs of the first, third, and fifth leaves from the shoot apex of several apple cultivars, including Jonathan, Fuji, Oorin, and Indo. In vitro-grown apple shoots of susceptible cultivars were then treated with various doses of X- or γ-ray irradiation. Several mutants resistant to AM-toxin I were obtained by combining the techniques for tissue culture of apple shoots with the AM-toxin I screening method. Following a repeat second screening test with AM-toxin I, mutant plants were sprayed with a spore suspension of A. alternata and found resistant to be the fungal pathogen. These mutants showed normal phenotypic appearance, and so far, no difference has been observed between the original plants and mutants except for the susceptibility to Alternaria blotch.     

ICChI, a glycosylated chitinase from the latex of Ipomoea carnea

A multi-functional enzyme ICChI with chitinase/lysozyme/exochitinase activity from the latex of Ipomoea carnea subsp. fistulosa was purified to homogeneity using ammonium sulphate precipitation, hydrophobic interaction and size exclusion chromatography. The enzyme is glycosylated (14–15%), has a molecular mass of 34.94 kDa (MALDI–TOF) and an isoelectric point of pH 5.3. The enzyme is stable in pH range 5.0–9.0, 80 °C and the optimal activity is observed at pH 6.0 and 60 °C. Using p-nitrophenyl-N-acetyl-β-d-glucosaminide, the kinetic parameters K_m, V_max, K_cat and specificity constant of the enzyme were calculated as 0.5 mM, 2.5 × 10⁻⁸ mol min⁻¹ μg enzyme⁻¹, 29.0 s⁻¹ and 58.0 mM⁻¹ s⁻¹ respectively. The extinction coefficient was estimated as 20.56 M⁻¹ cm⁻¹. The protein contains eight tryptophan, 20 tyrosine and six cysteine residues forming three disulfide bridges. The polyclonal antibodies raised and immunodiffusion suggests that the antigenic determinants of ICChI are unique. The first fifteen N-terminal residues G–E–I–A–I–Y–W–G–Q–N–G–G–E–G–S exhibited considerable similarity to other known chitinases. Owing to these unique properties the reported enzyme would find applications in agricultural, pharmaceutical, biomedical and biotechnological fields.     

In vitro Antagonism of Rhizobacteria Isolated from Coffea arabica L. against Emerging Fungal Coffee Pathogens

In vitro antagonistic effects of rhizobacteria associated with Coffea arabica L. against some fungal coffee pathogens were studied. The aims were to screen indigenous coffee‐associated isolates for their inherent antagonistic potential against major coffee wilt diseases induced by Fusarium spp. Antagonistic effects, siderophore, HCN and lytic enzyme production were determined on standard solid media. Chemical methods were employed to categorize the major types of siderophores. From a total of 212 rhizobacterial isolates tested, over 10 % (all Pseudomonas and Bacillus spp.) exhibited remarkable inhibition against Fusarium spp. One isolate AUPB24 (P. chlororaphis) showed maximum inhibition of mycelial growth against all fungal pathogens tested, whereas other isolates were mostly inhibitory to F. stilboides and F. oxysporum. The isolate AUBB20 (B. subtilis) was most antagonistic to F. xylarioides. Of the rhizobacterial isolates tested, 67 % produced siderophores and 35 % produced HCN. Many strains (all Pseudomonas spp.) produced siderophores of the hydroxamate type and only a small proportion produced those of the catecholate type. Few antagonists showed chitinase activity. The production of siderophores and HCN by Pseudomonas spp., lipase and protease by all antagonists and β‐1,3‐glucanase by several Bacillus spp. could be considered the major mechanisms involved in the inhibition of fungal growth. The in vitro results provide the first evidence of an antagonistic effect of coffee‐associated rhizobacteria against the emerging fungal coffee pathogens F. stilboides and F. xylarioides and indicate the potential of both bacterial groups for biological control of coffee wilt diseases.     

Predatory activity and passage of six nematophagous fungi species in gastrointestinal tract of trichostrongylide-infected sheep*

With the development of anthelmintic resistance of parasitic nematodes, screening the potential of predatory fungi candidates by in vitro and in vivo assay is necessary for biocontrol of parasitic nematodes in sheep. Fifteen native isolates of fungi species of six predators were evaluated through in vitro and in vivo experiment to evaluate the capacity of passing through the gastrointestinal tract of sheep. The results of the in vitro experiment showed that the reduction percentages of the third-stage larvae (L3) of trichostrongylides in faeces were 97.02–98.49% for five isolates of Arthrobotrys (Monacrosporium) sinense; 83.47–99.12% for three isolates of Arthrobotrys oviformis; 80.00–97.41% for four isolates of Arthrobotrys conoides; and 99.18%, 77.56%, and 75.72% for one isolate of Arthrobotrys microscaphoides, Arthrobotrys salina, and Dactylellina leptospora, respectively. In the in vivo assay, the results exhibited that the larval recoveries in faeces were significantly decreased (p?D. leptospora. After fungal treatment (within 24?h) of one isolate of A. salina, although the efficacy against L3 was 43.83%, tested fungus was detected in the faeces. The remaining isolates, except one isolate of D. leptospora and one isolate of A. conoides, were positive for either L3 reduction or fungal survival in faeces. The present study showed that nematophagous fungi could survive in the passage through the alimentary tract of sheep and should be potential candidates as a feed supplement.     

Photosynthetic responses to elevated CO2and O3 in field-grown potato(Solanum tuberosum)

Potato plants (Solanum tuberosum L. cv. Bintje) were grown to maturity in open-top chambers under three carbon dioxide (CO₂; ambient and 24 h d⁻¹ seasonal mean concentrations of 550 and 680 μmol mol⁻¹) and two ozone levels (O₃; ambient and an 8 h d⁻¹ seasonal mean of 50 nmol mol⁻¹). Chlorophyll content, photosynthetic characteristics, and stomatal responses were determined to test the hypothesis that elevated atmospheric CO₂ may alleviate the damaging influence of O₃ by reducing uptake by the leaves. Elevated O₃ had no detectable effect on photosynthetic characteristics, leaf conductance, or chlorophyll content, but did reduce SPAD values for leaf 15, the youngest leaf examined. Elevated CO₂ also reduced SPAD values for leaf 15, but not for older leaves; destructive analysis confirmed that chlorophyll content was decreased. Leaf conductance was generally reduced by elevated CO₂, and declined with time in the youngest leaves examined, as did assimilation rate (A). A generally increased under elevated CO₂, particularly in the older leaves during the latter stages of the season, thereby increasing instantaneous transpiration efficiency. Exposure to elevated CO₂ and/or O₃ had no detectable effect on dark-adapted fluorescence, although the values decreased with time. Analysis of the relationships between assimilation rate and intercellular CO₂ concentration and photosynthetically active photon flux density showed there was initially little treatment effect on CO₂-saturated assimilation rates for leaf 15. However, the values for plants grown under 550 μmol mol⁻¹ CO₂ were subsequently greater than in the ambient and 680 μmol mol⁻¹ treatments, although the beneficial influence of the former treatment declined sharply towards the end of the season. Light-saturated assimilation was consistently greater under elevated CO₂, but decreased with time in all treatments. The values decreased sharply when leaves grown under elevated CO₂ were measured under ambient CO₂, but increased when leaves grown under ambient CO₂ were examined under elevated CO₂. The results obtained indicate that, although elevated CO₂ initially increased assimilation and growth, these beneficial effects were not necessarily sustained to maturity as a result of photosynthetic acclimation and the induction of earlier senescence.     

Efficient Plant Regeneration System From Leaf Discs of Zonal (Pelargonium x hortorum) and two scented (P. capitatum and P. graveolens) Geraniums

An efficient and rapid plant regeneration system was established for zonal and scented geraniums using leaf discs as explants. Several explants, medium and culture conditions were studied to optimize shoot induction. Leaf discs taken from 4–5 weeks old in vitro grown plants, whatever the genotype, were more effective for shoot regeneration than those taken from greenhouse grown plants. Darkness proved to be a stimulating factor for shoot regeneration and the combination between NAA and two cytokinins gave the best results. Direct shoot regeneration (100%) was obtained from leaf discs of P. capitatum on half-strength MS medium supplemented with 0.5 mg l⁻¹ NAA in combination with 1 mg l⁻¹ of BAP and zeatin in darkness (11.4 shoots per explant). In the same medium and culture conditions, all P. graveolens leaf discs also exhibited direct shoot regeneration (7.3 shoots per explant). For P. x hortorum, 100% of leaf discs underwent shoot regeneration on a MS medium supplemented with 0.2 mg l⁻¹ NAA in combination with 0.5 mg l⁻¹ of BAP and zeatin in darkness (8.8 shoots per explant) or under low light conditions with 0.2 mg l⁻¹ NAA and 1 mg l⁻¹ of BAP and zeatin (7.5 shoots per explant). For this species, the best results for shoot elongation were obtained on half-strength MS medium gelled with Phytagel 0.3% (v/v). Whatever the genotype, all shoots rooted readily when transferred to diluted MS medium (MS/2) containing 1 mg l⁻¹ IAA. Acclimatized plants grew normally and flowered in greenhouse conditions. Flow cytometry analysis made on leaves of acclimatized plants revealed that all the scented geranium plants are similar to mother plants while 71% of P. x hortorum plants which showed strong growth were tetraploid.