Molecular study of phytoplasmas associated with pistachio yellows in Iran

A large‐scale survey was conducted on pistachio plants exhibiting foliar symptoms including scorch, little leaf, yellows and reddish in pistachio growing areas in the Qom, Yazd and Qazvin provinces of Iran. Total DNA was extracted from symptomatic and symptomless pistachio and used in nested PCR assays with phytoplasma universal primers. Nested PCR products were obtained for symptomatic plant samples while the symptomless plants yielded no PCR products. Virtual restriction fragment length polymorphism, phylogenetic and DNA homology analyses of partial 16S ribosomal sequences of phytoplasma strains associated with symptomatic plants revealed the presence of phytoplasmas referable to two ribosomal groups; in particular, “Candidatus Phytoplasma solani” and “Ca. P. phoenicium” were identified. The presence of these phytoplasmas in pistachio is of great phytosanitary significance due to its commercial interest.     

Detection of Aflatoxin in Aspergillus Species Isolated from Pistachio in Iran

To estimate the incidence contamination of fresh pistachio nuts by aflatoxigenic fungi in Iran, nut samples were collected from pistachio orchards in Kerman, Rafsanjan and Isfahan regions. Out of the 200 Aspergillus isolates obtained, 11 species were identified as A. alliaceous, A. candidus, A. flavus, A. niger, A. niveus, A. ochraceus, A. parasiticus, A. tamari, A. terreus, A. unguis and A. wentii. For detection of aflatoxin production ability of the isolates, three target genes, namely aflR, aflJ, and omtB, used in PCR amplification. In all the examined cases, the degenerate primer designed for amplification of omtB gene, named omtBII, was able to amplify an expected 611 bp fragment in aflatoxigenic isolates in this study and yielded the same result as those obtained from TLC analysis and fluorescence ability by application of methylated β‐cyclodextrin in culture media. Using this procedure the significant incidence of aflatoxin‐producing aspergilli was confirmed in pistachio nuts produced in different regions of Iran. The results indicated that PCR method described here, in combination with fluorescence assay, is a reliable and simple confirmatory test for monitoring pistachio nuts contaminated with aflatoxinogenic aspergilli.     

Isolation of Eutypa lata from Juglans regia, and Pathogenicity Studies on Different Wood Plants

The fungus Eutypa lata was isolated from diseased walnut trees (Juglans regia) exhibiting small cankers. The morphological characteristics of the culture and the pathogenicity were compared with those of known isolates of the fungus from other hosts. Inoculation tests with walnut isolates on grape, walnut, almond and apricot yielded characteristic cankers. Furthermore, pathogenicity tests on walnut with isolates from other different hosts resulted in differences in virulence. Similar differences in virulence were observed between 13 single ascospore isolates of the fungus ex apricot inoculated on walnut.     

Evaluation of Pochonia chlamydosporia var. chlamydosporia as a control agent of Meloidogyne javanica on pistachio

The potential of isolates of Pochonia chlamydosporia var. chlamydosporia as biocontrol agents for root-knot nematodes was investigated in vitro and on pistachio plants. On potato dextrose agar, growth of all isolates started at temperatures above 10°C, reached maximum between 25 and 28°C and slowed down at 33°C. On water agar, all isolates parasitized more than 85% of the eggs of Meloidogyne javanica at 18°C after 3 weeks. Filtrates of isolates grown on malt extract broth did not cause more than 5% mortality on second-stage juveniles of M. javanica after 48 h of incubation. A single application of 10×10³ chlamydospores (produced on sand–barley medium) g^–1 soil, was applied to unsterilised soil planted with pistachio cv. Kalehghochi, and plants were inoculated with 3000 nematode eggs. After 120 days in the glasshouse, nematode multiplication and damage were measured. Ability of fungus isolates to survive in the soil and to grow on roots were estimated by counting colony forming units (cfu) on semi-selective medium. Fungal abundance in soil increased nearly 3-fold and 10×10³ and 20×10³ cfu g^–1 root of pistachio were estimated in pots treated with isolates 40 and 50, respectively. Strain 50 was more abundant in soil and on the roots, infected more eggs (40%) on the roots and controlled 56% of total population of M. javanica on pistachio roots, whereas isolate 40 parasitized 15% of the eggs on the roots and controlled ca. 36% of the final nematode population.     

Distribution of Catenaria anguillulae Sorokin,a facultative endoparasite of nematodes in soils from different locations of India

A survey of soils from 39 different locations from 12 states of India for the presence/absence of Catenaria anguillulae indicated a wide distribution of the fungus in Indian soils. Out of 490 soil samples, 451 yielded C. anguillulae. Further, it was also noted that the fungus is present throughout the year in soils from the different locations irrespective of soil types and crops grown. This is the first report of this kind on distribution of C. anguillulae from India.     

Abundance and biological parameters of psyllophagous coccinellids in pistachio orchards

The abundance of coccinellids on pistachio trees was monitored at both a cultivated pistachio plantation and a wild pistachio growing area in Rafsanjan, Iran. Eleven ladybird species were identified from planted pistachio trees with Adalia bipunctata (Linnaeus) and Oenopia conglobata contaminata (Menetries) the most abundant ladybirds. At the wild pistachio plantation site, 17 coccinellid species were collected, with Brumus undulatus Weise the most abundant ladybird, followed by A. bipunctata and O. conglobata contaminata. Three species of Coccinellidae were evaluated for their potential for biological control of the common pistachio psylla (CPP), Agonoscena pistaciae Burckhardt and Lauterer. Coccinella undecimpunctata aegyptica (Reiche), Exochomus nigripennis (Erichson) and Hippodamia variegata (Goeze) developed successfully on A. pistaciae nymphs and reproduced successfully. Development and reproduction of these ladybirds were also examined on an alternative prey, the cotton aphid Aphis gossypii Glover, the most abundant aphid species on herbal weeds in pistachio orchards. The highest intrinsic rate of natural increase (r_m value) was observed in C. undecimpunctata aegyptica, 0.22 and 0.23 when it was fed on A. pistaciae and A. gossypii Glover, respectively. In comparison, the lowest r_m values (0.12 and 0.13) were found in E. nigripennis on the CPP and cotton aphid diets, respectively. Exochomus nigripennis consumed the largest number of psyllid nymphs during larval period, while the adults consumed the lowest number of psyllid nymphs daily. Although eight psyllophagous coccinellid species were collected in colonies of the CPP on pistachio trees, they did not regulate psyllid numbers below damaging levels. At present, conservation should assume a central role in biological control strategies for this pest.     

Biological characteristics ofCryptosporiopsis abietina on Hinoki cypress and its antagonistic effect to other microorganisms

In isolation tests on different parts of sound Hinoki cypress (Chamaecyparis obtusa), the coelomyceteCryptosporiopsis abietina was isolated, mostly from immature cone scales and inner bark areas of branches and main stems. In isolation tests using branches or stems of 3-yr-old seedlings to 35-yr-old adult cypresses from various localities,C. abietina was isolated in 28 to 100% of the cypresses tested. It was also confirmed that the fungus rarely infects seeds of the cypress, and can survive in dried seeds for several years, though it is not a seed-borne fungus. No symptoms appeared in inoculation tests with the fungus on 8-yr-old cypress. In inoculation test in a greenhouse, reliable infection without symptoms was obtained on wounded cypress seedlings. These results reveal thatC. abietina is a common endophytic fungus in Hinoki cypress. Confrontation and volatile antagonistic tests in Petri dishes indicated that some isolates ofC. abietina produce antifungal substances in vitro. On these isolates, the reproduction of a mycophagous nematodeBursaphelenchus xylophilus was retarded. The results of inoculation tests suggest that the possibility ofC. abietina as a primary causal agent of the resinous stem canker of Hinoki cypress is low. The role ofC. abietina in the cypress is briefly discussed.     

Molecular characterization of a seed transmitted clavicipitaceous fungus occurring on dicotyledoneous plants (Convolvulaceae)

Ergoline alkaloids (syn. ergot alkaloids) are constituents of clavicipitaceous fungi (Ascomycota) and of one particular dicotyledonous plant family, the Convolvulaceae. While the biology of fungal ergoline alkaloids is rather well understood, the evolutionary and biosynthetic origin of ergoline alkaloids within the family Convolvulaceae is unknown. To investigate the possible origin of ergoline alkaloids from a plant-associated fungus, 12 endophytic fungi and one epibiotic fungus were isolated from an ergoline alkaloid-containing Convolvulaceae plant, Ipomoea asarifolia Roem. & Schult. Phylogenetic trees constructed from 18S rDNA genes as well as internal transcribed spacer (ITS) revealed that the epibiotic fungus belongs to the family Clavicipitaceae (Ascomycota) whereas none of the endophytic fungi does. In vitro and in vivo cultivation on intact plants gave no evidence that the endophytic fungi are responsible for the accumulation of ergoline alkaloids in I. asarifolia whereas the epibiotic clavicipitaceous fungus very likely is equipped with the genetic material to synthesize these compounds. This fungus resisted in vitro and in vivo cultivation and is seed transmitted. Several observations strongly indicate that this plant-associated fungus and its hitherto unidentified relatives occurring on different Convolvulaceae plants are responsible for the isolated occurrence of ergoline alkaloids in Convolvulaceae. This is the first report of an ergot alkaloid producing clavicipitaceous fungus associated with a dicotyledonous plant.Data deposition: The sequences reported in this paper have been deposited in the GenBank (accession numbers are given in the text and in Fig. 3) Dedicated to Dr. Dr. h. c. mult. Albert Hofmann, the great pioneer of ergot research, on the occasion of his 100th birthday     

Secondary metabolites from endophytic fungi isolated from the Chilean gymnosperm Prumnopitys andina (Lleuque)

Two endophytic fungi isolated from the phloem of the Chilean gymnosperm Prumnopitys andinawere cultured in liquid potato-dextrose medium. The secondary metabolites were isolated and identified by spectroscopic methods. The fungus E-3 which could not be identified yielded 4-(2-hydroxyethyl)phenol, p-hydroxybenzaldehyde and the isochromanone mellein. The second fungus, identified as Penicillium janczewskii K.M. Zalessky yielded peniprequinolone and gliovictin, reported for the first time for this species. This is the first report on secondary metabolites produced by endophytic fungi from Chilean gymnosperms.     

First Report on Mould and Mycotoxin Contamination of Pistachios Sampled in Algeria

Mycotoxin contamination of pistachios represents a serious food safety hazard. The aim of this study was to evaluate fungal contamination and aflatoxin (AF) and ochratoxin A (OTA) occurrence in pistachio sampled in Algeria and to study the mycotoxigenic capacities of the isolates. A total of 31 pistachio samples were collected from retail outlets from different regions of Algeria. The most frequently found fungi were Penicillium spp. (38%), Aspergillus section Nigri (30%) and A. flavus (22%). A total of 56.5% of A. flavus isolates were able to produce AFB₁ and AFB₂. No A. section Nigri uniseriate isolate was OTA producer, whereas OTA production capacity was detected in 33.3% of the A. section Nigri biseriate. At least one of the potentially ochratoxigenic species was found in 64.5% of samples. Despite the high number of pistachio samples containing AFs and OTA-producing isolates, only two samples contained AFs (always below the EU maximum tolerable level) and only one sample showed OTA contamination. This is the first report on the occurrence of toxigenic moulds and mycotoxins in pistachios from Algerian market.     

Pollen effects on fruit and seed characteristics in pistachio (Pistacia vera L.)

The agronomic profitability of nut production in pistachio (Pistacia vera L.) is hampered by several factors such as the production of empty nuts, the small size and dry weight of the edible kernel and the low percentage of endocarp dehiscence. The causes underlining these problems are still unknown. The effect of pollinations with pollen from P. atlantica and six different P. vera genotypes on several fruit, nut and embryo characters was compared to study possible xenic and/or metaxenic effects. The use of P. atlantica pollen affected some traits, but there were no clear differences when the pollen of the different P. vera genotypes was used suggesting that, at the intraspecific level, the main limitation to pistachio fruit production problems might not be due to pollen factors.     

Genetic diversity of Ophiocordyceps sinensis, a medicinal fungus endemic to the Tibetan Plateau: Implications for its evolution and conservation

Background  

Ophiocordyceps sinensis (syn. Cordyceps sinensis), endemic to alpine regions on the Tibetan plateau, is one of the most valuable medicinal fungi in the world. Huge commercial demand has led to excessive harvest and a dramatic decline in its numbers. The diversity of terrains and climates on the Tibetan Plateau and the broad insect host range (more than 50 species in the family Hepialidae) may have resulted in substantial intraspecific genetic diversity for this fungus. The objective of this study was to evaluate the population distribution of O. sinensis from geographically diverse regions of the Tibetan Plateau based on nrDNA ITS and MAT1-2-1 gene sequences. Understanding of the genetic diversity and genesis of O. sinensis will provide important information for the evolution and conservation of this fungus.     

Separated components of root exudate and cytosol stimulate different morphologically identifiable types of branching responses by arbuscular mycorrhizal fungi

Two morphologically distinct hyphal branching responses by the AM fungus, Glomus intraradices, were stimulated by separated components of carrot root exudate. Complex branching up to the sixth order was induced by compounds most soluble in 35 % methanol, whereas the formation of more lateral branches (second order) was stimulated by compounds most soluble in 70 % methanol. This same 70 % alcohol soluble fraction also stimulated a completely different type of branching pattern in another fungus, Gigaspora gigantea. This pattern consisted of a very periodic distribution of dense clusters of hyphal branches that had a very high degree of complexity. In contrast to exudate components, separated cytosolic components of carrot roots did not stimulate any of the observed hyphal branching patterns. Alcohol-soluble fractions actually inhibited hyphal tip growth of G. gigantea and induced the formation of “recovery” branches that were identical to those induced by an inhibitor found in the exudate of Chard (Beta vulgaris ssp. cicla), a non-host plant.     

Four new coprophilous hyphomycetes

Four new and interesting coprophilous Hyphomycetes are described from India: (1)Sympodina coprophila gen. et sp. n. isolated from goat dung from Jaipur; this fungus produces 2-celled, brown, solitary, dry blastospores developing acrogenously at the tips of conidiophores and at their sympodially produced successive growing points; (2)Adhogamina ruchira gen. et sp. n. isolated from pony dung from Rishikesh; the fungus produces one-celled, brown, solitary, dry blastospores without longitudinal germ slit developing in basipetal order on conidiophores; (3)Bahupaathra samala gen. et sp. n. occurring on cow dung from Chakrata; the fungus produces distinct, erect, intricately branched conidiophores with fertile and sterile branches and one-celled slimy phialospores forming globose masses at the tips of characteristic collarettes; and (4)Angulimaya sundara gen. et sp. n. occurring on cow dung from Chakrata; the fungus produces erect conidiophores bearing crowded clusters of annellophores from which one-celled conidia are produced in simple dry basipetal chains. The forms are compared with related genera.     

Somatic hybrids between Solanum bulbocastanum and potato: a new source of resistance to late blight

 Solanum bulbocastanum, a wild, diploid (2n=2x=24) Mexican species, is highly resistant to Phytophthora infestans, the fungus that causes late blight of potato. However this 1 EBN species is virtually impossible to cross directly with potato. PEG-mediated fusion of leaf cells of S. bulbocastanum PI 245310 and the tetraploid potato line S. tuberosum PI 203900 (2n=4x=48) yielded hexaploid (2n= 6x=72) somatic hybrids that retained the high resistance of the S. bulbocastanum parent. RFLP and RAPD analyses confirmed the hybridity of the materials. Four of the somatic hybrids were crossed with potato cultivars Katahdin or Atlantic. The BC₁ progeny segregated for resistance to the US8 genotype (A-2 mating type) of P. Infestans. Resistant BC₁ lines crossed with susceptible cultivars again yielded populations that segregated for resistance to the fungus. In a 1996 field-plot in Wisconsin, to which no fungicide was applied, two of the BC₁ lines, from two different somatic hybrids, yielded 1.36 and 1.32 kg/plant under a severe late-blight epidemic. In contrast, under these same conditions the cultivar Russet Burbank yielded only 0.86 kg/plant. These results indicate that effective resistance to the late-blight fungus in a sexually incompatible Solanum species can be transferred into potato breeding lines by somatic hybridization and that this resistance can then be further transmitted into potato breeding lines by sexual crossing. Received: 27 October 1997 / Accepted: 11 November 1997     

Natural Occurrence of Seiridium cardinale on Thuja in Germany

Seiridium cardinale (Wagener) Sutton & Gibson (syn. Coryneum cardinale Wagener) is the most dangerous parasitic fungus of cypress and other Gupressaceae, already appearing in epidemic proportions, especially in the mediterranean area. Recently (in 1989, 1990 and 1991) this pathogen has been detected in the northern part of Germany (Hamburg and Schleswig-Holstein) naturally infecting Thuja. The geographical distribution and the progressive spread of S. cardinale is reviewed. Diagnostic features of the fungus, modes of host penetration, principal symptoms of the disease, ways of dispersal and control methods are described. Possible causal factors for the recently observed occurrence of 5. cardinale in the north of Germany are discussed.     

Increased Levels of Cytokinins in Barley Leaves Having the Systemic Acquired Resistance to Bipolaris sorokiniana (Sacc.) Shoemaker

The role of endogenously induced higher level of cytokinins and exogenously applied kinetin in relation to the development of barley leaf spot caused by Bipolaris sorokiniana (Sacc.) Shoemaker (syn. Helminthosporium sativum Pammel, King and Bakke) was studied. Spraying barley leaves with kinetin suppressed the number and the size of necrotic spots caused by the fungus. Inoculation of the lower leaves of barley by a spore suspension of the fungus B. sorokiniana induced resistance on the upper leaves against a subsequent challenge inoculation by the same pathogen 10 days later. An increase in the level of cytokinins was observed in these resistant leaves. Elevated levels of cytokinins may cause a type of juvenility in leaf tissues. The juvenile state could be in a causal relationship with the suppression of necrotic spots caused by the fungus.     

Reaction of some tomato cultivars to tomato leaf curl virus and evaluation of the endophytic colonisation with Beauveria bassiana on the disease incidence and its vector,Bemisia tabaci

Tomato line LA1478 and Pusa Ruby were resistant to tomato leaf curl virus (TLCV) disease. They registered higher plant height, number of branches, total phenol content and yield per plant than the other cultivars. Variety Peto 86 was tolerant to the disease while the other popular tomato cultivars, i.e. Ace, Early Pack, Money Maker, Prichard and Strain B were highly susceptible to the disease. Plant height and number of branches per plant revealed significantly positive association with fruit yield per plant. The disease index of TLCV exhibited significant negative correlations with plant height, total phenol content and fruit yield per plant – 0–4 and 5–25 adult whiteflies were observed on resistant susceptible cultivars. In the case of epiphytically colonisation by Beauveria bassiana conidia, not all developing hyphae on the leaf surface penetrated the whitefly cuticle. Many of the germ tubes elongated to a short distance before terminating its growth. On the other hand, the rapid staining of tomato tissues injected with B. bassiana conidial suspension indicates that the entomopathogenic fungus was established inside tomato tissues until the end time of the trial. The direct injection with the spore suspension yielded high post-colonisation, where the fungus was recovered from sites distant from the point of inoculation. This indicates that the fungus has the potential to move throughout the plant tissues. Laboratory bioassay of tomato whitefly feeding on tomato tissues containing B. bassiana conidial spores indicates that plant endophytic colonisation with entomopathogenic fungi may reduce insect survival on these plants. LT₅₀ values of the test diet were between three and four days. The mortality of Bemisia tabaci was high in the case of endophytically colonisation compared to epiphytically one (90.0% compared to 10.0% during three days) for whiteflies fed tomato tissues containing 1.5 × 10⁷ B. bassiana spores/ml. Application of B. bassiana as an artificial endophyte inside tomato plants can be an important component in the integrated control of tomato whiteflies. The endophytic colonising can achieve biocontrol effect based on induced disease resistance in plant tissues. According the available references, this is the first report on B. tabaci controlling by plant endophytic treatment.     

Enhancement of biosurfactants and biofilm production after gamma irradiation-induced mutagenesis of Bacillus subtilis UTB1, a biocontrol agent of Aspergillus flavus

Bacillus subtilis UTB1 is known as a biocontrol agent of Aspergillus flavus in pistachio nuts. In order to reduce growth of this fungal pathogen to a greater extent, a random mutagenesis using gamma irradiation was applied in strain UTB1. We studied the effects of different doses of irradiation (from 100 Gray to 3000 Gray) and efficiency of 500 selected colonies was assessed against A. flavus in a plate assay. Forty-five colonies exhibited higher inhibition activity compared to the non-irradiated wild type. Eight mutants out of the 45 were selected based on different polymorphism patterns obtained by rep-PCR (ERIC and BOX). Six mutants demonstrated enhanced production of biosurfactants on blood agar medium and in oil spreading technique and they also revealed more robust biofilm in comparison with the wild type UTB1. These observations showed that the six mutants are more effective biocontrol agents than the parental strain, suggesting that they would be promising biocontrol candidates against A. flavus in pistachio nuts.     

DNA cloning and screening of a partial genomic library from an arbuscular mycorrhizal fungus,Scutellospora castanea

A technique has been developed to efficiently extract purified, restrictable genomic DNA from spores of different arbuscular mycorrhizal fungi in order to begin detailed investigations of the genome of the Glomales. The protocol yielded variable amounts of DNA depending on the fungal species; for Scutellospora castanea and Gigaspora rosea it reached values of 1.5–2 ng/spore. EcoRI digests of DNA from S. castanea were cloned into pUC18 and about 1000 recombinant DNA clones were obtained. Of those screened, 50 contained inserts of 500–7000 bp. Selected inserts detected DNA sequences from S. castanea spores or roots infected by this fungus, but not from nonmycorrhizal roots. This is the first report of a partial genomic library from an arbuscular mycorrhizal fungus.