Studies on the pycnidial formation by macrophomina phaseoli (Maubl.) ashby size of pycnidia,ostiole and pycnidiospores and its significance

Summary Studies on the size of pycnidia, ostiole, length and width of pycnidiospores and ratio between length and width of pycnidiospores ofM. phaseoli isolates from cotton, castor, sesame and groundnut, the pycnidia of which were obtained by artificial inoculation on the hosts and also in culture medium of one cotton isolate, revealed that all the isolates were true to the type species and that the isolates had significant differences among them for these characters. The measurements of pycnidia and spores were dependant on the substratum for their production, and as such should preferably be made when these structures are obtained on the host of the isolate. Delimitation ofM. phaseoli on basis of these measurements from other hosts of the isolates is considered unwarranted or invalid.     

Interactions of soil fungi with Macrophomina phaseoli (Maubl.) ashby,the cause of root rot of cotton

Summary Interactions of 46 isolates of fungi with an isolate ofM. phaseoli from cotton, was studied in agar culture. They were grouped into 4 different types of reactions.T. viride was found to inhibit the growth ofM. phaseoli and grow over its colony. The hyphae ofT. viride were observed to coil around the hyphae ofM. phaseoli. A majority of the isolates tested had no effect on the growth ofM. phaseoli. M. phaseoli, however, was found to overgrow the test fungi.     

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.     

Inhibition of fungi,actinomycetes and bacteria by Stachybotrys atra

The effect ofStachybotrys atra on fungi, actinomycetes and bacteria was studied in agar culture. Of the 27 genera and 52 species of fungi, 6 species of actinomycetes and 5 genera and 10 species of bacteria tested,S. atra was found to inhibit 95.7 % fungi, all the actinomycetes and 83.3 % of bacteria.Aspergillus ustus, 2 unidentified species ofFusarium, Rhizobium trifolii andPseudomonas spp. were however not inhibited. In experiments in soil,S. atra inhibited the growth ofTrichoderma viride. S atra was ineffective whereT. viride andS. atra were inoculated simultaneously or whereT. viride was added 7 days earlier. Besides,S. atra was not found to controlMacrophomina phaseoli infection on cotton. Soil culture ofS. atra showed phytotoxic effect on cotton seedlings.S. atra produced a dark brown to black thermostable toxic metabolite in the medium. The culture filtrate retarded the growth ofM. phaseoli in agar culture. Despite the importance ofS. atra as a fungistatic agent with wide antimicrobial spectrum, the use ofS. atra as a protectant against plant pathogens seems to be limited because of its phytotoxicity on cotton.     

Effects of temperature,light, carbon and nitrogen nutrition on reproduction in Phoma medicaginis

Reproduction by three isolates ofPhoma medicaginis growing on potato dextrose agar was studied. The formation of pycnidia was optimum at 30°C whereas the optimum for the formation of conidia was 20°C. Light consistently increased the numbers of pycnidia and conidia over those formed in darkness and more conidia were produced in light at 23°C than at 30°C. The effects of carbon and nitrogen sources on reproduction were studied using modified Richard's medium as the basal medium. Fourteen carbohydrates, 11 amino acids, 9 inorganic nitrogen sources and urea were evaluated by replacing the carbohydrate or nitrogen in Richard's medium with the test substance. Generally, the monosaccharides and disaccharides were about alike and superior to polysaccharides for the production of pycnidia. The carbon sources were about equally useful for production of conidia, but the polysaccharides were superior to the other two classes of carbon sources when the number of conidia/pycnidium was calculated. Generally, the formation of pycnidia and conidia was favored by nitrate more than by ammonium nitrogen sources. The average number of conidia/pycnidium was greatest, however, when the nitrogen source was NH₄NO₃. All amino acids tested appeared to be useful nitrogen sources for production of pycnidia but none were especially good for conidia production. L-isoleucine was superior to the other amino acids tested. Of three isolates used, Illinois 23 consistently produced more pycnidia and conidia that did isolates Minnesota 2 and Missouri 5. Usually the significant interactions between isolates and other treatments were due to a greater response by isolate Ill. 23. It was concluded that the reproduction ofP. medicaginis varies significantly with the isolates of the fungus, the environment, and nutrients as well as with interactions among these factors, and conclusions about the influence of a particular factor will depend on whether the formation of pycnidia, conidia or conidia/pycnidium is being studied.Paper No. 7425, Scientific Journal Series, Minnesota Agricultural Experiment Station.     

Survival of Macrophomina phaseoli (Maubl.) Ashby on cucurbit roots

Summary Experiments on the survival ofM. phaseoli on cucurbit root pieces were carried out. The sclerotia ofM. phaseoli were almost unaffected by 10 months dry storage in the laboratory. Under dry to moderately moist soil conditions it survived for 2 months with little loss of viability. In wet soils however, the buried root pieces were colonized by other soil microorganisms which prevented the growth ofM. phaseoli.     

Physiologic specialisation in Macrophomina phaseoli (Maubl.) Ashby., causing stem rot of jute,corchorus species

With eight isolates ofMacrophomina phaseoli, pathogenicity test was conducted on twenty varieties of jute belonging to two species,Corchorus capsularis andC. olitorius. Against six isolates the varieties screened showed different degrees of disease reactions while against the remaining two, all the varieties gave similar resistant reaction. As differential varieties, a minimum of four recognised the seven physiological races ofMacrophomina phaseoli. From the cultural characteristics, however, all the eight isolates differed from each other and were fitted in a dichotomous key as eight cultural races.

---

Zusammenfassung Mit acht Stämmen vonMacrophomina phaseoli sind Pathogenitätstest an zwanzig Varietäten von Jute durchgeführt worden, die zwei Arten:Corchorus capsularis undC. olitorius angehören. Gegen sechs Stämme zeigten die untersuchten Varietäten einen verschiedenen Grad der krankhaften Reaktionen, während gegen die restlichen zwei Stämme alle Varietäten gleichartige Widerstandsfähigkeit aufwiesen. Mindestens vier von den sieben physiologischen Rassen vonM. phaseoli sind erkannt worden. Betreffs der Kulturcharakteristik wichen alle acht Stämme von einander ab und sind als echte Kulturrassen beschrieben.

     

Influence of culture media and environmental factors on mycelial growth and pycnidial production of Sphaeropsis pyriputrescens

Sphaeropsis pyriputrescens, the causal agent of Sphaeropsis rot of pears and apples, is a recently described species. In this study the effects of culture media, temperature, water potential, pH and light on mycelial growth and pycnidial production of S. pyriputrescens were evaluated. Apple juice agar and pear juice agar were most suitable for mycelial growth of all six isolates tested. Cornmeal agar was not suitable for either mycelial growth or pycnidial production. The fungus grew from -3 to 25 C, with optimum growth at 20 C and no growth at 30 C. The fungus grew at water potential as low as -5.6 MPa on potassium chloride-amended potato-dextrose agar (PDA). Hyphal extension was not observed at -7.3 MPa after 10 d incubation, but growth resumed when the inoculum plugs were placed on PDA. The fungus grew at pH 3.3-6.3 and optimum growth was at pH 3.3-4.2. No mycelial growth was observed at pH above 7.2 after 10 d incubation, but growth resumed when the inoculum plugs were transferred onto PDA. Regardless of medium tested, few pycnidia formed at 20 C in the dark. Pycnidial production was enhanced significantly by fluorescent light, but continuous light appeared to reduce pycnidial production, depending on the medium. Oatmeal agar (OMA) was most suitable for production of pycnidia and conidia. Pycnidia that formed on 3 wk old OMA cultures at 20 C under 12 h light/12 h dark produced abundant conidia, and the technique is recommended for inoculum production.     

Sulphur and phosphorus requirements of three fungi causing diseases in storage

Summary The effect of different sulphur and phosphorus compounds on the growth and reproduction of three fungi causing storage rot, viz.,Fusarium solani, Botryodiplodia ananassae andMacrophomina phaseoli has been studied. Sixteen different sources of sulphur were used and out of them magnesium sulphate was found to be most favourable for the growth and reproduction of all the three fungi. Sodium sulphite and sodium bisulphite were toxic. Potassium metabisulphite prevented growth ofF. solani and M. phaseoli while it supported moderate growth ofB. ananassae. Only magnesium sulphate could induce the sporulation ofB. ananassae while sporulation and sclerotial development ofF. solani andM. phaseoli respectively varied with the type of sulphur sources used. Optimum concentration of magnesium sulphate was also determined and it was found that the growth and sporulation ofF. solani andB. ananassae were best at 0.375 g/l and 0.75 g/l.M. phaseoli tolerated higher doses of this substance as the best growth and excellent sclerotial development were recorded at 3.0 g/l (the maximum concentration used). Phosphorus was found to be essential for the present fungi as none of them could grow in complete absence of this substance. Onthophosphates and nucleic acid, were found to be favourable sources for growth and reproduction of the 3 organisms.     

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.     

Detection,identification and morphological characteristic of Macrophomina phaseolina: the charcoal rot disease pathogens isolated from infected plants in Northern Jordan

This is the first report about charcoal rot disease in Jordan. Twenty-five Macrophomina phaseolina isolates were collected from infected plants showing typical symptoms of charcoal rot disease. All of the 25 M. phaseolina isolates were pathogenic to cucumber plants under green house effect. The amplification of the isolated DNA from the 25 pathogenic fungal cultures using ITS specific primers (ITS 1?+?ITS 4) showed a single band of 580?bp. There was a significant variation of their mycelial linear growth rate on PDA medium. The 25 M. phaseolina isolates showed a wide heterogeneity in their mycelium colour, microsclerotia distribution, pycnidia formation and chlorate phenotypes. Based on the morphological characterisation, the 25 isolates were grouped into seven different groups as indicated in a dendrogram of their morphological variation. The overall means similarity matrix of the 25 M. phaseolina recovered isolates were 0.58. The means of similarity matrix of the 25 M. phaseolina was in between 0.83 and 0.14. The similarity coefficient between the 25 isolates varies between 0.27 and 1.0.     

Utilization of oligosaccharides by three pathogenic isolates of colletotrichum gloeosporioides

Summary Utilization of six oligosaccharides (viz., sucrose, maltose, cellobiose, lactose, raffinose and melezitose) by the isolates ofColletotrichum gloeosporioides Penz. causing leaf-spot diseases ofCarissa carandas L.,Eucalyptus robusta Sm. andBougainvillaea glabra Choizy has been studied. Daily chromatographic analysis of the culture medium revealed that the utilization of sucrose, maltose, cellobiose and raffinose was through a hydrolytic pathway. In case of lactose and melezitose no hydrolytic product could be detected. During the utilization of sucrose two oligosaccharides (Rf 0.27 and 0.2) made their appearance. TheBougainvillaea-isolate synthesized two oligosaccharides (Rf 0.18 and 0.1) in a maltose medium although no glucose could be traced, while in case of the other two isolates, in addition to these synthetic oligosaccharides, glucose also appeared. When theCarissa-isolate ofColletotrichum gloeosporioides was grown on a cellobiose medium, in addition to glucose, two synthetic oligosaccharides (Rf 0.15 and 0.11) were formed. Other two isolates showed the formation of one oligosaccharide (Rf 0.15) and no glucose. The dry weight of mycelium recorded an increase on oligosaccharides with slow rate of consumption, while on oligosaccharides with rapid rate of utilization it tended to become constant or showed a decline towards the end of the incubation.     

Morphology of the fruiting pustules in the genus Pestalotia

Summary The morphology of fruiting pustules in six isolates ofPestalotia causing the leaf spot diseases ofSyzygium cumini, Eugenia heyneana, Carissa congesta, Madhuca indica, Mangifera indica andPsidium guajava was studied. On the first two hosts both pycnidial as well as acervulus like fruiting bodies were produced. OnMadhuca indica the structure of fructifications varied on the two surfaces of the leaf while on the remaining hosts, the fructifications were typically acervular in nature. Considerable fluctuations in the structure of the fruiting bodies were observed.     

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.     

Expression of elastinolytic activity among isolates inAspergillus SectionFlavi

A survey of the distribution of elastinolytic potential among 32 culture collection isolates ofAspergillus flavus, A. oryzae, A. parasiticus, A. sojae, A. nomius, andA. tamarii revealed this character to be highly conserved withinAspergillus SectionFlavi. Furthermore, 144 isolates ofA. flavus from environmental samples from six separate regions of the United States produced elastase on solid medium. Most previously described polymorphisms in elastinolytic potential were attributed to the toxicity of borate buffers. Replacement of borate with HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) resulted in detection of elastase production on solid medium by all tested fungal isolates except two that had been in culture over 50 years. In liquid culture, only isolates ofA. flavus, A. tamarii, andA. oryzae accumulated elastase activity. Although isoelectric focusing revealed only one isoform (pI 9.0) of elastase in these culture filtrates, elastinolytic activity in filtrates was partially inhibited by both 1,10-phenanthrolene (2 mM) and phenylmethylsulfonylfluoride (2 mM), suggesting the presence of both metallo and serine elastinolytic proteinases.Abbreviation IEF isoelectric focusing The US Government's right to retain a non-exclusive, royalty-free licence to any copyright is acknowledged.     

Biological control of white rot of onion

Summary Interactions of 123 isolates of fungi, 17 of bacteria and 22 of actinomycetes, respectively, withSclerotium cepivorum were studied in agar culture. They were grouped into 4 different types of reactions. Amongst themTrichoderma viride, Fusarium graminearum, Coniothyrium minitans andGliocladium roseum inhibited the growth ofS. cepivorum and later grew over its colony.T. viride showed a characteristic coiling around the hyphae ofS. cepivorum. T. viride andF. graminearum prevented the development of sclerotia.C. minitans was found to parasitize the sclerotia ofS. cepivorum and produced its pycnidia within them.Aleurisma carnis, Cladosporium elatum, Penicillium expansum, P. nigricans, P. notatum, P. piscarium, P. puberulum, P. rolfsii, P. urticae, P. variabile, Tilachlidium humicola andHelminthosporium sp. inhibited the growth ofS. cepivorum at a distance. Eleven isolates of bacteria and 3 ofStreptomyces sp. showed pronounced antagonistic properties againstS. cepivorum.Experiments were carried out to study the effects on white rot development in soil of organisms selected from agar plate tests. None of the antagonistic micro organisms had any deleterious effects on onion growth. Of the organisms testedP. nigricans gave the best results in controlling white rot infection.This work was carried out at the Department of Botany, The University, Birmingham, England. I wish to thank Prof.C. J. Hickman for his invaluable advice and encouragement.     

Aflatoxin B1 producing potential of isolates of Aspergillus flavus Link ex Fries from cotton,maize and wheat

Twenty-one isolates ofAspergillus flavus Link ex Fries obtained from cotton, maize and wheat were screened for their ability to produce aflatoxins on two liquid media. Of these, sixteen isolates were toxigenic and produced only aflatoxin B₁ as assessed by bioassay on okra seedlings and TLC method. For screening isolates ofA. flavus for aflatoxin formation, 0.7 % YES+ Salt medium was found to be good as also for obtaining higher yields of the toxin. Isolates ofA. flavus produced aflatoxin B₁ ranging from 0.85 to 17.2 mg/50 ml. Maximum yield of aflatoxin was obtained when rice was used as the substrate in case of toxigenic isolates L-27 and C-9, and on maize in isolate M-11.     

Morphological and molecular identification of Leptosphaeria maculans in canola seeds and flowers collected from the North Iran

Blackleg disease, caused by Leptosphaeria maculans, is one of the most important diseases of rapeseed Brassica napus in Iran as in other regions of the world. The samples including canola petals and seeds were collected during 2014–2015 from canola field in North Iran. Isolates characteristics of fungus were assessed based on the colony growth rate and pycnidia in Potato Dextrose Agar. The pycnidia of the fungus were black, globose to subglobose in shape, the single-celled conidia, hyaline and fusiform with diameters of 4–5 × 1.5–2 μm. Most of the isolates were produced pigment in the liquid culture in variable color brown to black. Thirteen isolates were then separated into pathogenicity groups based on the interactions on B. napus differential cultivars. For the direct detection of seed contamination with L. maculans, PCR was developed using specific primers pair (LmacF, LmacR) which can amplify ITS1 and ITS2 along with the 5.8S rRNA region of L. maculans genome. Based on the followed information and sequence analysis, the fungal isolates from these samples were identified as L. maculans. The findings of this research showed that the disease was aggressive and highly distributed from infested seeds to oilseed rape fields.     

Effect of seed and root exudates on the growth ofXanthomonas phaseoli var.fuscans

In a medium containing bean, barley and wheat seed exudates,Xanthomonas phaseoli var.fuscans (Burk.) Starr et Burk. grew substantially better than in that containing root exudates of these plants. When the bacteria were cultivated in a medium containing root exudates of bean plants deprived of cotyledons after eleven days of growth, growth was slower than in the presence of root exudates of control plants. On the other hand, the growth was stimulated in a medium containing root exudates of bean plants deprived of leaves. It was found that seed exudates of these plants contained biologically active peptides stimulating the growth of the microorganism. These peptides were not found in root exudates. These findings suggest a relationship between the survival ofXanthomonas phaseoli var.fuscans in the rhizosphere of bean and the exudation of biologically active peptides originating from the stock substances of seeds and cotyledons.     

In vitro propagation ofMedicago andLotus species by node culture

Summary Nodes ofMedicago sativa, Lotus corniculatus, Lotus tenuis, andLotus pedunculatus were cultured on MS basal media with different growth regulators. InM. sativa each node produced one shoot and the apical dominance was unaffected by high levels of cytokinins, and subsequent cycles of culture. Shoot development was stimulated by the presence ofN ⁶-isopentenyl-adenine in the culture medium and was dependent on the genotype of the explant. Shoot development was not affected by the original position of the node on the plant nor by the plant age. Shoots rooted in MS medium gelled with starch and containing 2 mg·liter⁻¹ indol-3-acetic acid. In the threeLotus species, node culture was a more effective technique than inM. sativa. The number of shoots per node increased with the time of culture and with the presence of 0.05 mg·liter⁻¹ of 6-benzylaminopurine. The highest number of shoots derived from one node was achieved inL. pedunculatus and inL. tenuis by culturing single nodes, whereas inL. corniculatus stem segments had to be totally covered by the medium for success. Rooting was easily achieved in MS medium with or without auxins.