Factors affecting sporulation by Phomopsis phaeolorum

Light and nutrition are the important factors in the production of pycnidia and conidia by cowpea isolates of the Phomopsis state of Diaporthe phaseolorum. The highest number of pycnidia and conidia were produced on plant tissue exposed to cool-white fluorescent light. In semisynthetic media more pycnidia were formed at high glucose concentrations, but they matured more slowly than those formed at lower glucose concentrations. Both the level of conidiation and the percentage of pycnidia that formed conidia were higher at lower glucose concentrations. The best artificial medium for inducing a high number of pycnidia containing abundant conidia was one that contained 0.4% glucose and 0.4% NaNO₃. A number of carbon sources could replace glucose in this medium.     

Effect of culture media, temperature and light on radial growth and pycnidium production of cowpea isolates of Phoma bakeriana

The effects of culture media, temperature and light on radial growth and pycnidium production of cowpea isolates (PB1, PB2, PB3) of Phoma bakeriana were investigated in vitro. There was no isolate effect on growth and pycnidium production but some evidence of a medium effect. Exceptionally high growth rate on two media corresponded with high pycnidium production; while low growth rates on other media were generally associated with high sporulation. There was a large temperature effect and an isolate effect on growth with a significant temperature by isolate interaction. Temperatures of 5°C, 10°C and 35°C inhibited pycnidium formation completely while the range 15–30°C had no effect on sporulation. Maximum growth and pycnidium production occurred at 25°C. Near-ultraviolet radiation seemed to have caused higher pycnidium counts than the other light sources.     

Submerged culture conidial germination and conidiation of the bioherbicideColletotrichum truncatum are influenced by the amino acid composition of the medium

Summary Submerged culture experiments were conducted to determine the optimal nitrogen source for rapidly producing conidia of the bioherbicide,Colletotrichum truncatum. Germination ofC. truncatum conidial inocula in submerged culture occurred most rapidly (>95% in 6 h) in media provided with a complete complement of amino acids. When (NH₄)₂SO₄, urea, or individual amino acids were provided as the sole nitrogen source, conidial germination was less than 20% after 6 h incubation. Conidia production was delayed inC. truncatum cultures grown in media with urea or individual amino acids as nitrogen sources compared to cultures supplied with Casamino acids or complete synthetic amino acid nitrogen sources. The use of methionine, lysine, tryptophan, isoleucine, leucine or cysteine as a sole nitrogen source severely inhibitedC. truncatum conidia production. Media with synthetic amino acid mixtures less these inhibitory amino acids produced significantly higher conidia yields compared to media with amino acid mixtures containing these amino acids. When various amounts of each individual inhibitory amino acid were added to media which contained amino acid mixtures, cysteine and methionine were shown to be most effective in reducing conidiation. An optimal nitrogen source forC. truncatum conidiation in submerged culture should contain a complete mixture of amino acids with low levels of cysteine, methionine, leucine, isoleucine, lysine and tryptophan for rapid conidiation and optimal conidia yield.The mention of firm names or trade products does not imply that they are endorsed or recommended by the US Department of Agriculture over other firms or similar products not mentioned.     

Growth and glucoamylase production by the thermophilic fungusThermomyces lanuginosus in a synthetic medium

Summary The production of glucogenic amylase from the thermophilic fungus Thermomyces lanuginosus was studied in shake flasks and laboratory fermentors. As conidia were not able to germinate in media without yeast extract, pregerminated conidia were applied as inoculum. By this procedure it was possible to use different NH _inf4 ^sup+ salts as the sole source of nitrogen for growth and amylase formation in a synthetic medium. In pH-controlled fermentors a fourfold increase in the extracellular glucogenic amylase activity was obtained with (NH₄)H₂PO₄ as the nitrogen source as compared with yeast extract. However, by fractionation of these activities, comparable yields of partially purified glucoamylases were obtained. The glucoamylase preparation from fermentations with either of the nitrogen sources had a temperature optimum at 70° C and showed similar thermal stability. By incubation without substrate at 60° C. 90% of the activity was still present after 5 h. At 70° C, 50% of the activity was retained after 30 min incubation. Offprint requests to: I. Hassum     

Effect of light intensity on sporulation of Botryosphaeria ribis

Pycnidia were produced by six of seven isolates ofB. ribis at one or more intensity levels of continuous illumination at 21 °C. Under conditions of alternating light (12 h–27 °C) and darkness (12 h–21 °C) pycnidia formed in cultures of six isolates at three or more intensity levels, while one isolate failed to form pycnidia at any intensity level. Pycnidia did not develop when cultures were incubated in complete darkness. Exposure periods as brief as 2 days under continuous illumination at 21 °C induced pycnidial formation. In alternating light (12 h–27 °C) and darkness (12 h–21 °C), the shortest period of exposure which induced pycnidial formation was 4 days. Continuous illumination at 21 °C favored development of uniloculate pycnidia, while alternating light (12 h–27 °C) and darkness (12 h–21 °C) favored formation of multiloculate pycnidia.Contribution No. 22 from The Botany Section of The Department of Biology, The Pennsylvania State University.     

Occurrence of Phomopsis longicolla β Conidia in Naturally Infected Soybean

Although Phomopsis longicolla is primarily known as a seedborne pathogen, it can be isolated from all parts of the plant. The disease lesions observed on the basal parts of soybean stems were slightly sunken with irregular shapes and sizes, bordered by a thin black margin. Within the lesions themselves, large and diffusely distributed pycnidia with α and β conidia, typical of the genus Phomopsis, were observed. The percentages of the two types of conidia varied considerably, but β conidia were predominant in most of the pycnidia. The presence of these reproductive organs indicated that the symptoms could have been caused by Phomopsis sojae. However, after isolation on a nutritive medium, all cultural and morphological characteristics clearly indicated that the isolated fungus was P. longicolla, whose identification was subsequently confirmed by sequencing three genomic regions. Monosporic isolates, with different ratios of α and β conidia, exhibited a high level of pathogenicity on soybean, after artificial inoculation. Both types of conidia were observed on the stems of the inoculated soybean plants. Beta conidia also formed quickly on medium made of soybean seeds and mature stems after exposure to low temperatures (?10°C). This study suggests that P. longicolla is capable of a massive production of β conidia, not only in old fungal cultures as it had until now been believed, but also in infected soybean plants in the field.     

Certain chemical requirements for growth and sporulation of alternaria species

Summary Fresh, single-spore isolates ofAlternaria nyctanthi spec. nov. andAlternaria tropaeoli Deshpande &N. R. Rajderkar from the infected leaves varied widely in response when cultured on Coon's, PDA, Richard's and Czapek's medium. Some isolates did not produce macroscopically visible colonies, although the spores germinated. A few isolates grew poorly without sporulating; most of the others grew well and sporulated abundantly on PDA and Czapek-Dox medium.Cultures that had been inhibited from sporulating by irradiation at 26° C sporulated when subsequently placed in a 22° C incubator, even though nitrogen availability was varied by adding solutions of NaNO₃, with or without sucrose.     

Phenotypic characteristics of root-nodulating bacteria isolated from Acacia spp. grown in Libya

Thirty isolates of root-nodulating bacteria obtained from Acacia cyanophylla, A. karroo, A. cyclops, A. tortilis (subsp.raddiana), Faidherbia albida and Acacia sp., grown in different regions of Libya, were studied by performing numerical analysis of 104 characteristics. Three fast- and one slow-growing reference strains from herbaceous and woody legumes were included. Five distinct clusters were formed. The fast-growing reference strains were separated from the isolates whereas the slow-growing was included in cluster 4. With the exception of one cluster, the majority of clusters were formed regardless of the host plant or site of origin. Based on plant tests, generation times, acid production and carbon utilization the isolates were diverse (fast and slow-growing isolates). Like slow-growing isolates, most of the fast-growing isolates appeared to be non-specific, nodulated many species from the same genus notably F. albida, known to nodulate only with slow-growing strains. Most clusters grew at temperatures 35 °C and 37 °C; some grew at temperatures above 40 °C. The majority of isolates grew at acid and alkaline pH and only one isolate grew below pH 4. Most isolates were able to utilize many amino acids as nitrogen sources and to reduce nitrate. Urea was hydrolysed by all clusters. Monosaccharides and polyols were used by slow and fast-growing isolates as the only carbon sources whereas assimilation of disaccharides varied: Some isolates, like slow-growing isolates, failed to utilize these carbon sources. Most isolates were unable to utilize polysaccharides. Regarding tolerance to NaCl on agar medium, the majority of isolates were unable to grow at a concentration of 2% NaCl, but some were highly resistant and there was one isolate which grew at 8% NaCl. Most isolates were resistant to heavy metals and to antibiotics.     

Effect of temperature and sources of carbon and nitrogen on the conidial germination and appresoria formation in Colletotrichum capsici

The effect of temperatures and exogenous supply of different carbon and nitrogen sources on the conidial germination and appresoria formation inC. capsici has been studied. 25 °C was observed to be the best temperature for conidial germination. At 18 °C, conidia germinated only in hanging drops and did not germinate on 2 % agar. Amongst carbon sources, 1-rhamnose supported maximum conidial germination and appresoria formation. Potassium nitrate supported very good conidial germination and appresoria formation. Ammonium nitrate, ammonium sulphate and ammonium chloride were found to be inhibitory. A few aminoacids stimulated conidial germination but dl-alanine, dl-methionine, tyrosine and l-glutamine were found to be inhibitory.     

Influence of Nitrogen Source, Thiamine, and Light on Biosynthesis of Abscisic Acid by Cercospora rosicola Passerini

Abscisic acid production by Cercospora rosicola Passerini in liquid shake culture was measured with different amino acids in combination and singly as nitrogen sources and with different amounts of thiamine in the media. Production of abscisic acid was highest with aspartic acid-glutamic acid and aspartic acid-glutamic acid-serine mixtures as nitrogen sources. Single amino acids that supported the highest production of abscisic acid were asparagine and monosodium glutamate. Thiamine was important for abscisic acid production. Leucine inhibited abscisic acid production. C. rosicola produced abscisic acid in the dark, but production more than doubled in the presence of light.     

Effect of Nutrition on Growth and Sporangial Production of Two West African Isolates of Phytophthora palmivora Butl

Nutrition of two isolates of Phytophthora palmivora was studiedin pure culture. Several amino acids supported good growth ofboth isolates but casein hydrolysate was the best nitrogen sourcefor one isolate. Ammonium sulphate, urea and potassium nitratewere poor nitrogen sources for both isolates. Growth of bothisolates was enhanced by ferric iron in the presence of L-ascorbicacid. Calcium chloride was stimulatory to one isolate whilecholestrol and calcium chloride enhanced the growth of bothisolates. Sporangial production varied in both isolates on mediacontaining various amino acids.     

Vapor Fixation of Intractable Fungal Cells for Simple and Versatile Scanning Electron Microscopy

Imaging of exudation of conidia from fruiting bodies by scanning electron microscopy is challenging because of the difficulty in achieving specimen fixation that enables conidia to be immobilized from fruiting bodies. Conidia in chains borne on conidiophores are readily dissipated on coming in contact with liquid fixatives before fixation becomes effective. Two phialide‐forming fungi including Aspergillus and Penicillium species, and a pycnidium‐forming fungus Fusicoccum species were subjected to a vapor fixation procedure for scanning electron microscopy rather than conventional immersion fixation procedures. The intractable fungi were exposed to the vapor of 2% glutaraldehyde and 2% osmium tetroxide each for at least 2 h, subsequently followed by gold coating. Through the vapor fixation, excellent retention of conidial chains in phialides was achieved in this study. It appeared that conidial masses from pycnidia were usually held together in globose to oval drops. The simple vapor fixation procedure for preserving delicate fungal cells could be widely used for routine applications to facilitate morphological characterization of diverse plant pathogenic fungi as they are in ex planta ecological niches during the saprophytic phase as well as in host parts during the parasitic phase.     

Involvement of Hydrogen Peroxide, Peroxidase and Superoxide Dismutase in Response of Medicago truncatula Lines Differing in Susceptibility to Phoma medicaginis Infection

This study was undertaken to assess the involvement of hydrogen peroxide (H₂O₂), peroxidase (POX; EC 1.11.1.7) and superoxide dismutase (SOD; EC 1.15.1.1) in Medicago truncatula in relation with susceptibility to Phoma medicaginis infection. Several M. truncatula lines were studied in terms of their response to P. medicaginis infection. Fifteen days after inoculation (dai), differences in susceptibility were found. DZA45.5 was the least susceptible line and F83005.5 was the most susceptible line. Microscopic analysis of fungal development was performed in inoculated detached leaves of the DZA45.5 and F83005.5 lines. No significant difference was observed in events from conidia germination to penetration. Differences became apparent during the colonization process as the pathogen was able to sporulate rapidly increasing its concentration on the tissue of F83005.5 in comparison with DZA45.5. To characterize the susceptibility of the two lines, histochemical detection of H₂O₂ was made in detached leaves. H₂O₂ detection showed an early accumulation of this component in cells of DZA45.5 at 1 dai. However, H₂O₂ was detected in few, if any, cells in the tissues of the most susceptible line, F83005.5. The activity of POX and SOD were determined spectrophotometrically in leaves of intact inoculated plants of both lines. Phoma medicaginis inoculation of DZA45.5 and F83005.5 did not affect POX activity level in leaves when compared with control uninoculated plants. SOD activity showed a significant decrease in F83005.5 and DZA45.5 leaves at 4 dai and 9 dai, respectively, in comparison with control plants. In control plants POX activity was significantly higher in the least susceptible line DZA45.5 in comparison with F83005.5. Early and higher production of H₂O₂ and elevated basal POX activity in cells of the least susceptible line, DZA45.5 could explain its ability to be less favourable to the colonization and reproduction of P. medicaginis in comparison with the most susceptible line, F83005.5.     

Variation in nitrogen source utilisation by Pisolithus isolates maintained in axenic culture

The influence of various inorganic and organic nitrogen sources on biomass production by 17 isolates of Australian Pisolithus spp. was investigated before and after a 3-year maintenance period in axenic culture. While some isolates produced similar or higher amounts of biomass on NH₄ ⁺ or certain amino acids after the maintenance period, there was a general trend to reduced biomass production on these substrates. Biomass production by most isolates on bovine serum albumin increased significantly after maintenance. The data are discussed in relation to the use of axenic culture growth experiments for investigations of inter- and intraspecific physiological variation in ectomycorrhizal fungi. Accepted: 22 February 2001     

Phacidiopycnis washingtonensis--a new species associated with pome fruits from Washington State

A new species of Phacidiopycnis associated with pome fruits is described. The fungus causes fruit rot on apples during storage and is associated with a twig dieback and canker disease of crabapple trees and dead twigs of pear trees. To characterize the biology of the fungus and compare it with Ph. piri, the type species of the genus, effects of nine media and light on mycelial growth and pycnidial production, mycelial growth in response to temperature and mode of conidial germination in response to nutrient were determined. Apple-juice agar, pear-juice agar, prune-juice agar, potato-dextrose agar (PDA) and malt-extract agar, Czapek-Dox agar and oatmeal agar (OMA) favored mycelial growth. Cornmeal agar (CMA) did not favor mycelial growth. Light effect on pycnidial formation was medium dependent. Abundant pycnidia with mature conidia formed in 14 d old PDA and OMA cultures at 20 C, regardless of light, whereas none or very few pycnidia formed on other media in the dark. Fluorescent light stimulated formation of pycnidia except on CMA. The fungus grew at -3-25 C, with optimum growth at 15-20 C. Conidia germinated either by forming germ tubes or less often by budding. Budding of conidia occurred in 1 and 10% pear-juice solutions but not in 100% pear-juice solution. Six isolates of Ph. washingtonensis from different species of pome fruits had identical ITS sequences. The sizes of the ITS region were the same for both Ph. washingtonensis and Ph. piri, and four polymorphic nucleotide sites were found in the ITS region between Ph. washingtonensis and Ph. piri. The similarity in ITS sequences between these two taxa is confirmatory evidence for the erection of the new species of Phacidiopycnis associated with pome fruits we describe here.     

Studies on Aspergilli XVI. Effect of pH,temperature, and carbon and nitrogen interaction

Summary The effect of pH, temperature, and carbon and nitrogen interaction on the growth and sporulation ofAspergillus nidulans (Eidam)Wint.,A. rugulosus Thom &Raper,A. variecolor (Berk. &Br.)Thom &Raper andA. quadrilineatus was studied. All the moulds could grow on a wide range of pH (2.0 to 12.0) but the growth was poor on too acid and too alkaline media. Best growth ofA. rugulosus, A. quadrilineatus, andA. violaceus was seen at pH 6.5 and that ofA. nidulans andA. variecolor at pH 7.0. In general maximum production of perithecia was recorded between pH 6.0 and 8.0.All the above species ofAspergillus under study could grow between a temperature range of 10° C–48° C, but the growth was poor at 10° C and 48° C. The present moulds showed good growth at 20° C, 25°C, and 30° C. At 40° CA. nidulans andA. rugulosus showed moderate growth while the rest of the Aspergilli attained good growth. Temperatures between 20° C–30° C favoured excellent perithecial production.In general, little improvement in growth was noted on media containing good carbon and nitrogen sources. Malic acid was found to be useless when supplied singly. But, poor growth was recorded when supplied in combination with amino acids, amide, and peptone. This was due to the fact that these N sources also supplied carbon for their metabolism.     

Amine Oxidases of Microorganisms

The amine oxidase was found to be formed in mycelia of fungi when they were grown on monoamines or diamines as sole nitrogen sources. The maximal formation of enzyme was observed in the initial stage of growth, then the enzyme disappeared semilogarithmically. Other sources of nitrogen, such as ammonia, nitrate, urea and amino acids, were fully inactive for the enzyme formation. Furthermore, ammonia repressed the enzyme formation by fungi. The amine oxidase of fungi resembled in substrate specificity the monoamine oxidase of animal tissues. The enzyme oxidized preferentially aliphatic monoamines of C₃–C₆. Agmatine and histamine were also oxidized but in lower rates. Benzylamine was well oxidized by the enzymes of Aspergillus niger and Penicillium chrysogenum, but not by the enzymes of Monascus anka and Fusarium bulbigenum. Polyamines were not oxidized by the fungal enzymes.     

Studies on the nutrition of the genus pestalotiopsis

Four isolates ofPestalotiopsis, viz.P. gracilis, P. paeoniae isolates I and II, andP. royenae were isolated from the leaf spots ofAchras sapota, Anona squamosa, Syzygium cumini andCarissa congesta respectively. Studies on the nutrition of the isolates were carried out in liquid basal meduim in stationary cultures. Preliminary experiments had shown that a pH of 5.5 and a temperature of 25° C± 1° C were the most suitable for the growth of the present organisms. The growth response of the various species, as judged by their mycelial dry weight on 26 different carbon sources was studied.Among the pentoses, D-xylose supported poor growth of all the four isolates whereas L-arabinose and L-rhamnose were as good as glucose in efficiency, or occasionally better. Among the hexoses, D-fructose and D-mannose were superior to glucose (except forP. royenae). Galactose and L-sorbose were poor sources, the latter being poorest among all the carbohydrates used in this study. The disaccharides were generally superior to glucose except lactose which was poor. AlsoP. royenae had slighthly inferior growth on trehalose than on glucose. Maximum growth, among disaccharides, was attained on cellobiose. The nutritional value of trisaccharide, raffinose, was maximum for all butP. royenae which too grew better on it than on glucose. The three polysaccharides, viz. soluble starch, dextrin and inulin were much superior than glucose except forP. royenae. Generally sugar alcohols were equal to or better than glucose in efficiency. Glycerol, however, was a very poor source. The acids supported very poor growth of all the isolates.     

Effects of carbon and nitrogen sources,carbon-to-nitrogen ratio,and initial pH on the growth of nematophagous fungus <Emphasis Type="Italic">Pochonia chlamydosporia</Emphasis> in liquid culture

The effects of carbon and nitrogen sources, carbon-to-nitrogen ratio (C:N) and initial pH value on the growth and sporulation of the nematophagous fungus Pochonia chlamydosporia in liquid culture were examined. Among the 21 carbon sources and 15 nitrogen compounds tested, the optimal carbon and nitrogen sources for mycelial growth were sweet potato and L-tyrosine, and for sporulation were sweet potato and casein peptone. A C:N ratio of 10:1 at pH 3.7 gave the maximum yield of conidia and a C:N ratio of 40:1 at pH 6.8 gave the maximum biomass. The initial pH value had a significant effect on mycelial growth and conidial production, with the optimal ranges being 3.5–4.5 for sporulation and 5–6 for growth. Maximum conidial production was obtained at an initial pH of 4.0 and the maximum biomass at pH 6.0. The results also showed that the final pH after 7 days cultivation was always higher than the initial value. The variability in growth and sporulation of seven strains of P. chlamydosporia in liquid culture was also compared and discussed.