Nitrogen-metabolizing enzymes of Diplodia maydis, a Zea mays L. stalk rot causing fungus.

The nitrogen source available to Diplodia maydis in vivo is reported to affect the severity of stalk rot in maize. Nitrate and (or) ammonium salts were tested for their effect on the type of nitrogen metabolism found in Diplodia maydis in vitro. The level of glutamate dehydrogenase remained essentially constant on either nitrogen salt but nitrate reductase was induced by growth on nitrate salts and was not extractable on ammonium salts. Properties of nitrate reductase reported here are similar to those reported for the higher plant and Neurospora crassa enzymes. Thr relationship of nitrogen metabolism in Diplodia maydis to Zea mays L. stalk rot is discussed.     

DNA phylogeny, morphology and pathogenicity of Botryosphaeria species on grapevines

Several species of Botr yosphaeria are known to occur on grapevines, causing a wide range of disorders including bud mortality, dieback, brown wood streaking and bunch rot. In this study the 11 Botryosphaeria spp. associated with grapevines growing in various parts of the world, but primarily in South Africa, are distinguished based on morphology, DNA sequences (ITS-1, 5.8S, ITS-2 and EF1-α) and pathological data. Botryosphaeria australis, B. lutea, B. obtusa, B. parva, B. rhodina and a Diplodia sp. are confirmed from grapevines in South Africa, while Diplodia porosum, Fusicoccum viticlavatum and F. vitifusiforme are described as new. Although isolates of B. dothidea and B. stevensii are confirmed from grapevines in Portugal, neither of these species occurred in South Africa, nor were any isolates of B. ribis confirmed from grapevines. All grapevine isolates from Portugal, formerly presumed to be B. ribis, are identified as B. parva based on their EF1-α equence data. From artificial inoculations on grapevine shoots, we conclude that B. australis, B. parva, B. ribis and B. stevensii are more virulent than the other species studied. The Diplodia sp. collected from grapevine canes is morphologically similar but phylogenetically distinct from D. sarmentorum. Diplodia sarmentorum is confirmed as anamorph of Otthia spiraeae, the type species of the genus Otthia (Botryosphaeriaceae). A culture identified as O. spiraeae clustered within Botryosphaeria and thus is regarded as probable synonym. These findings confirm earlier suggestions that the generic concept of Botryosphaeria should be expanded to include genera with septate ascospores and Diplodia anamorphs.     

Phylogeny of the Botryosphaeriaceae reveals patterns of host association

Three anamorph genera of the Botryosphaeriaceae namely Diplodia, Lasiodiplodia and Dothiorella have typically dark, ovoid conidia with thick walls, and are consequently difficult to distinguish from each other. These genera are well-known pathogens of especially pine species. We generated a multiple gene genealogy to resolve the phylogenetic relationships of Botryosphaeriaceae with dark conidial anamorphs, and mapped host associations based on this phylogeny. The multiple gene genealogy separated Diplodia, Lasiodiplodia and Dothiorella and it revealed trends in the patterns of host association. The data set was expanded to include more lineages of the Botryosphaeriaceae, and included all isolates from different host species for which ITS sequence data are available. Results indicate that Diplodia species occur mainly on gymnosperms, with a few species on both gymnosperms and angiosperms. Lasiodiplodia species occur equally on both gymnosperms and angiosperms, Dothiorella species are restricted to angiosperms and Neofusicoccum species occur mainly on angiosperms with rare reports on Southern Hemisphere gymnosperms. Botryosphaeria species with Fusicoccum anamorphs occur mostly on angiosperms with rare reports on gymnosperms. Ancestral state reconstruction suggests that a putative ancestor of the Botryosphaeriaceae most likely evolved on the angiosperms. Another interesting observation was that both host generalist and specialist species were observed in all the lineages of the Botryosphaeriaceae, with little evidence of host associated co-evolution.     

Botryosphaeriaceae as potential pathogens of prunus species in South Africa, with descriptions of Diplodia africana and Lasiodiplodia plurivora sp. nov

Botryosphaeriaceae are common dieback and canker pathogens of woody host plants, including stone fruit trees. In the present study the diversity of members of the Botryosphaeriaceae isolated from symptomatic wood of Prunus species (plum, peach, nectarine and apricot) was determined in stone fruit-growing areas in South Africa. Morphological and cultural characteristics as well as DNA sequence data (5.8S rDNA, ITS-1, ITS-2 and EF-1a) were used to identify known members and describe novel members of Botryosphaeriaceae. From the total number of wood samples collected (258) 67 isolates of Botryosphaeriaceae were obtained, from which eight species were identified. All species were associated with wood necrosis. Diplodia seriata (= "Botryosphaeria" obtusa) was dominant, and present on all four Prunus species sampled, followed by Neofusicoccum vitifusiforme and N. australe. First reports from Prunus spp. include N. vitifusiforme, Dothiorella viticola and Diplodia pinea. This is also the first report of D. mutila from South Africa. Two species are newly described, namely Lasiodiplodia plurivora sp. nov. from P. salicina and Diplodia africana sp. nov. from P. persica. All species, except Dothiorella viticola, caused lesions on green nectarine and/or plum shoots in a detached shoot pathogenicity assay.     

High-resolution melting analysis: a new molecular approach for the early detection of Diplodia pinea in Austrian pine

The differentiation of Diplodia pinea from closely related species, such as Diplodia scrobiculata and Diplodia seriata, and its detection in plant tissue, represented a critical issue for a long time. Molecular screening tools have recently been developed to address this topic. In this study we applied one of the most sensitive and rapid diagnostic screening method so far developed, called High-Resolution Melting Analysis (HRMA), to detect D. pinea in Austrian pine (Pinus nigra). HRMA exploits differences in the melting behaviour of PCR products to rapidly identify DNA sequence variants without the need for cumbersome post-PCR methods. We developed a HRMA method to detect specific fungal sequences in the mitochondrial small subunit ribosome gene (mt SSU rDNA). The reliability of this technique was firstly assessed on DNA extracted from pure cultures of D. pinea and closely related species. Amplicon differences were screened by HRMA and the results confirmed by direct DNA sequencing. Subsequently, HRMA was tested on DNA from symptomatic and symptomless pine shoots, and the presence of the fungus was also confirmed by both conventional and molecular quantitative approaches. The HRMA allowed the distinction of D.?pinea from closely related species, showing specific melting profiles for the each pathogen. This new molecular technique, here tested in a plant-fungus pathosystem for the first time, was very reliable in both symptomatic and symptomless shoots. HRMA is therefore a highly effective and accurate technique that permits the rapid screening of pathogens in the host.     

Fungi on Acacia arabica Linn

Summary The paper deals with three new species of Deuteromycetous fungi e.g.Diplodia acaciae, Cytospora acaciae, andFusicoccum indicum collected on dead stems ofAcacia arabica Linn.     

Bioactivity studies of oxysporone and several derivatives

Recently oxysporone, a phytotoxic dihydrofuropyranone, was isolated along with two closely related compounds, afritoxinones A and B, from liquid cultures of Diplodia africana, an invasive fungal pathogen of Phoenicean juniper. In this study, eight derivatives were hemisynthesized and assayed for their phytotoxic and antifungal activities in comparison to the parent compound. Each compound was tested on non-host plants and on four destructive plant pathogens such as Athelia rolfsii, Diplodia corticola, Phytophthora cinnamomi and P. plurivora. The results on the phytotoxic activity showed that the dihydrofuropyranone carbon skeleton and both the double bond the hydroxy group of dihydropyran ring appeared to be structural features important in conferring activity. Although the data concerning the antifungal activity did not allow to extract any structure–activity relationships, it should be underlined that the conversion of oxysporone into the corresponding 4-O-benzoyl derivative led to a compound showing a good antifungal activity towards three out of the four organisms tested.     

6,7-Dimethoxycoumarin,a citrus phytoalexin conferring resistance against Phytophthora gummosis

6,7-Dimethoxycoumarin was isolated from the bark of citrus trunks, branches and fruit peels following inoculation with the fungus Phytophthora citrophthora. The compound inhibited growth in vitro of Phytophthora citrophthora, Verticillium dahliae, Penicillium digitatum, P. italicum, Colletotrichum gloeosporioides, Diplodia natalensis and Hendersonula toruloidea.     

Dry mass changes in germinating spores of Diplodia maydis

Summary The dry mass of two-celled Diplodia maydis spores was measured both before and after germination by quantitative interference microscopy. The dry mass of spores declined approximately 50% during germination. However, the dry mass of germinating spores plus the dry mass of their germ tubes was greater than the dry mass of spores before germination. We conclude that the germinating spores absorbed nutrients released from non-germinating spores.The dry mass of fungal spores can be estimated by weighing large numbers of spores and determining the mean from sample spore counts. Mumford and Pappelis(4) determined the total dry mass of individual spores of Fusarium roseum and the contained lipid bodies before and after spores germinated using quantitative interference microscopy. The mean spore dry mass before germination was 57 pg. Lipid bodies accounted for about 61% of that mass and decreased as spores germinated. The total dry mass of the spore and germ tube 24 hr later greatly exceeded that of the spore before germination. Quantitative interference microscopy has been used to measure the dry mass of various types of cells. Kulfinski and Pappelis (3) recently reviewed how this technique has been applied to plant cells. Technical aspects of interference microscopy have been described by Ross (6).The purpose of this study was to examine the dry mass changes in Diplodia maydis (Berk.) Sacc. with and without germ tubes through the use of interference microscopy.     

Stalk Rot of Maize: Host-pathogen Interaction

Four maize cultivars were each separately inoculated with 10 different isolates of Diplodia zeae, Fusarium moniliforme, F. graminearum and Macrophomina phaseolina. D. zeae consistently caused most stalk rot, followed in decreasing order by M. phaseolina, F. graminearum and F. moniliforme. Significant intraspecific differences in stalk rot were recorded. No significant stalk rot x cultivar interaction occurred. Stalk rot correlated non-significantly with yield loss but significantly with cellulase production in vitro.     

Morphological and molecular characterization of Diplodia seriata,the causal agent of canker and twig dieback disease on mulberry in Iran

Mulberries are cultivated for different purposes: for feeding larvae of silkworm, Bombyx mori, as fresh and dry food resources, in wood instrument industry, in pharmaceutical industry and as outdoor ornamental trees in Iran. In recent years, twig and branch canker disease symptoms have been noticed on mulberry trees in northwestern parts of Iran. Diplodia isolates were repeatedly recovered from symptomatic tissues. Based on cultural and morphological features, the isolates were identified as Diplodia seriata. The identity of the isolates was further confirmed using sequence data from internal transcribed spacer (ITS)-rDNA and ef-1α gene. A phylogenetic analysis using ITS sequence data clustered the isolates obtained in this study together with known Diplodia seriata isolates of other woody hosts from GenBank. Inoculation studies carried out on white mulberry twigs using an excised shoot method revealed that the isolates are pathogenic on this host. D. seriata have been reported from other woody host plant species such as Juglans nigra and Vitis vinifera in Iran, however, to the best of our knowledge, the occurrence of D. seriata on mulberry trees is new for Iran. The distribution and reaction of different Morus spp. to D. seriata remain to be studied.     

Mycotoxic peripheral myelinopathy, myopathy, and hepatitis caused by Diplodia maydis in vervet monkeys.

During recent historical times many Africans changed their diet to one based on maize. The grain is regularly contaminated by fungi which are toxigenic to domestic animals and birds. After one of the fungi, Diplodia maydis, in pure culture on maize, was added to the food of omnivorous primates there was demyelination of nerves, atrophy, degeneration and necrosis of muscle, and hepatitis. These preliminary results are applicable to veterinary and laboratory animal sciences. They may also be medically significant since neuromuscular syndromes of unknown cause are prevalent among Africans. Nerve conduction velocities and organ weights are defined for vervet monkeys.     

Induced sporulation of Botryodiplodia theobromae Pat. isolated from Araucaria cunninghamii Ait. in Malaysia

Summary Botryodiplodia theobromae Pat. isolated fromAraucaria cunninghamii Ait. in Malaysia was grown on a series of standard media and inoculated into both living and dead leaves ofAraucaria. A few pycnidiospores were produced in stromata on aged cultures on 2% malt medium whereas pycnidia were produced rapidly in living and dead leaves. Pycnospores were generally two-celled and either hyaline or dark brown and bore longitudinal striations. The validity of includingBotryodiplodia theobromae as a synonym ofDiplodia natalensis Pole Evans is discussed.     

Can the emergence of pine Diplodia shoot blight in France be explained by changes in pathogen pressure linked to climate change?

Sphaeropsis shoot blight, caused by Diplodia pinea and Diplodia scrobiculata, damage conifers throughout the world. In France, the first disease outbreaks were reported during the 1990s. The factors associated with the pathogen presence in stands and the relationship between pathogen and disease distributions were analysed in order to understand the Sphaeropsis emergence. Eighty‐two stands of Pinus nigra, Pinus sylvestris, Pinus pinaster and Pinus radiata were visited. Cones were collected on the ground to assess the pathogen frequency. Diplodia spp were isolated and determined by a species‐specific PCR test. The role of potential explaining factors of D. pinea prevalence on cones was analysed by logistic regression. D. pinea was the dominant species in visited stands. The main factors influencing the pathogen presence selected in the models were host species (the pathogen being less frequent on P. pinaster than on P. nigra and P. sylvestris cones), winter temperature and summer rain, which were both positively correlated with cone colonization. The climate became more favourable to D. pinea presence within the last 15 years compared with the previous 30‐year period. By contrast, future climatic changes over the next 40 years should have far less impact on the pathogen presence.     

Effect of Fungal Infection and Temperature on Physiological Changes in Banana Fruit

An attempt was made to investigate which factor temperature or fungal infection, has a greater effect in accelerating physiological processes such as respiration and ethylene evolution in fruit. For this study the effect of infection with Trichothecium roseum, Alternaria alternata and Diplodia natalensis on banana fruit, at 14°C and 20°C was examined. The results show that the development of rot caused by different fungi and the physiological changes in the infected fruit may depend not only on the effect of temperature on fungi (as in vitro) but also on fruit ripening, or on processes connected with fruit ripening, which may accelerate fungal development.     

金柚贮藏真菌病害及保鲜技术研究

1998年5月-2000年10月间调查了梅州柚果贮藏期的真菌病害,它们是:①柚果绿霉病(Penicilliumdigitatum),②柚果炭疽病(Collettrichumgloeosporioides),③柚果黑腐病(Alternariacitri),④柚果焦腐病(Diplodianatalensis)和⑤柚果酸腐病(Oosporacitri-aurantii)等5种.从几种安全性高的保鲜剂中筛选出1个混用配方,该配方对柚果贮藏防腐保鲜效果明显,贮藏5个月无病斑果达100%,6个月也只有20%的柚果出现少量病斑,且柚果几项质量指标较好.     

Toxicity of Diplodia maydis to laboratory animals.

Ten strains of Diplodia maydis isolated from commercial corn samples and grown on whole yellow corn at 25 degrees C for 6 weeks were toxic to ducklings and rats. The degree of toxicity depended on the incubation period and temperature. Minimum incubation periods of 3 to 4 weeks and 6 weeks were necessary to cause mortality in ducklings and rats, respectively. Cultures incubated at 31 and 25 degrees C were much more toxic than those kept at 16 and 20 degrees C. Heat treatments at 80 to 90 degrees C destroyed most of the toxicity of moldy meal. Mild lesions of a similar histopathological nature were found in subclinically and lethally poisoned rats. These included toxic myocarditis, enteritis, focal renal tubular necrosis, degeneration and peripheral necrosis of the islets of Langerhans, and a generalized venous congestion.     

Toxicity of Diplodia maydis to laboratory animals.

Ten strains of Diplodia maydis isolated from commercial corn samples and grown on whole yellow corn at 25 degrees C for 6 weeks were toxic to ducklings and rats. The degree of toxicity depended on the incubation period and temperature. Minimum incubation periods of 3 to 4 weeks and 6 weeks were necessary to cause mortality in ducklings and rats, respectively. Cultures incubated at 31 and 25 degrees C were much more toxic than those kept at 16 and 20 degrees C. Heat treatments at 80 to 90 degrees C destroyed most of the toxicity of moldy meal. Mild lesions of a similar histopathological nature were found in subclinically and lethally poisoned rats. These included toxic myocarditis, enteritis, focal renal tubular necrosis, degeneration and peripheral necrosis of the islets of Langerhans, and a generalized venous congestion.     

Two new species of Botryosphaeria with brown, 1-septate ascospores and Dothiorella anamorphs

Botryosphaeria sarmentorum sp. nov. and B. iberica sp. nov. are described and illustrated. These two species are unusual in this genus because of their brown, 1-septate ascospores. Phylogenetic analysis based on ITS and EF1-alpha sequences place them within the clade containing species with Fusicoccum anamorphs. The brown, 1-septate conidia, however, do not conform to Fusicoccum. Therefore phylogenetically and morphologically the anamorphs of these two species belong in a genus distinct from any of the currently accepted anamorph genera assigned to Botryosphaeria. Through a study of the type species of Dothiorella this genus is resurrected to accommodate anamorphs of Botryosphaeria with brown, 1-septate conidia. Botryosphaeria sarmentorum is shown to be the teleomorph of Diplodia sarmentorum, which in turn is transferred to Dothiorella. Otthia quercus is transferred to Botryosphaeria as B. quercicola nom. nov.     

Fungi associated with roots and fruits of black peppers in the Dominican Republic

Several soilborne diseases have occurred in black pepper (Piper nigrum) plantations in the Dominican Republic since 1977. To understand environments in which the disease occurs fungal floras associated with them were studied. A total of 1,119 isolates, 603 from root segments of 12 vines, and 516 from fruit samples of 30 vines were identified as belonging to a total of 39 genera, namely, 31 from roots and 26 from fruits, excluding unsporulated fungi. The most dominant fungi wereFusarium in both root and fruit samples, followed byRhizoctonia, Diplodia, andPythium from roots, andDiplodia, Colletotrichum andPestalotia from the fruits. Preliminary pathogenicity tests towards leaves or roots of black pepper were conducted.