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.     

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.

     

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.     

Pathogenicity of Macrophomina phaseoli on Jute in the Presence of Meloidogyne incognita and Hoplolaimus indicus

Seedlings of Corchorus capsularis (cv. C4444) were inoculated with Meloidogyne incognita, Hoplolaimus indicus, and a fungus pathogen of jute, Macrophomina phaseoli, separately and in all possible combinations. The significant damage of jute plants caused individually by the pathogens was aggravated when the fungus was associated with either of the nematode species. M. incognita alone caused greater damage than either H. indicus or Macrophomina phaseoli alone. Plants inoculated with M. incognita and Macrophomina phaseoli were more severely damaged than plants inoculated with H. indicus and the fungus. Plant growth was minimum and disease symptoms were maximum when all pathogens acted together. In the presence of the fungus, M. incognita produced fewer galls. The reproduction of H. indicus was not influenced by the other organisms.     

Possibility of using a bacterial antagonist against fungal diseases of Piper betle L. and corchorus spp.

Summary A strain ofBacillus subtilis isolated from soil was found effective againstSclerotium rolfsii when grown on 20% potato extract for 48 hours and used along with the medium. Whole boiled potato was also a good medium but the bacteria needed 20 days to grow until insoluble starch was digested.In field trials the antagonist brought down the spread ofSclerotium and anthracnose diseases ofPiper betle. Against seed and air-borne diseases of jute caused byMacrophomina phaseoli,Diplodia corchori andColletotrichum corchori, the antagonist also showed promising results.Concluding part of a scheme supported by Food & Agriculture Council, Pakistan.     

Studies on the pycnidial formation by Macrophomina phaseoli (Maubl.) Ashby. Development of pycnidia and pycnidiospores

Summary An account on the pycnidial development of eight isolates ofM. phaseoli — five from cotton and the other, one each from castor, groundnut and sesame and also from a culture medium has been given. The pycnidial development ofM. phaseoli from cotton on a culture medium is extremely rare and pycnidia developed were having the outer wall consisting of only one layer of thickened cells and no clear ostiole. In the other isolates, all from the hosts, there were no differences in the development of pycnidia and pycnidiospores. All the isolates were true to the type speciesM. phaseoli.     

Pectinolytic enzymes associated with the soft rots ofCitrus sinensis caused byAspergillus aculeatus andBotryodiplodia theobromae

Botryodiplodia theobromae andAspergillus aculeatus causing soft rots ofCitrus fruits were grown on wheat offal medium. The filtered washings of the wheat offal cultures were then assayed and found to contain pectinmethylesterases (PME) and polygalacturonases (PG). The two pectinases were also detected in large quantities from the filtrates of rotted fruits ofCitrus sinensis incited by each of the two test fungi. Two enzyme filtrates, one from rotted fruits ofCitrus sinensis and the other made up of pectinases from fungal cultures were introduced onto sterilised degreened fruits. Each of the two filtrates induced rot symptoms similar to those incited by the test fungi.Aspergillus aculeatus produced less PME and more PG thanBotryodiplodia theobromae.     

Characterization of an extracellular endopolygalacturonase from the saprobe Mucor ramosissimus Samutsevitsch and its action as trigger of defensive response in tropical plants

In recent years, interest in the ability of non-pathogenic microorganisms to induce resistance in plants has grown, particularly with respect to their use as environmentally safe controllers of plant disease. In this study, we investigated the capacity of Mucor ramosissimus Samutsevitsch to release pectinases able to degrade cell walls of Palicourea marcgravii St. Hil., a tropical forest native Rubiaceae on which the spores of this saprobic fungus have been found. The fungus was grown in liquid culture medium containing pectin as the sole carbon source and filtrates were analyzed for pectinase activity. An endopolygalacturonase was partially purified by ion exchange chromatography, gel filtration, and preparative isoelectrofocusing, and characterized. This enzyme was more active upon pectic substrates with a low degree of methyl esterification. The products of hydrolysis of different pectic substrates (including pectin from P. marcgravii) by the action of this endopolygalacturonase elicited to different extents the phytoalexin production in soybean cotyledons. Also, the enzyme itself and the products of its action on the pectic fraction of P. marcgravii elicited the production of defensive compounds in the leaves of the plant. These results suggest that, besides the role in recycling organic matter, saprobes may also play an important role in the induction of defensive mechanisms in wild plants by enhancing their non-specific resistance against pathogens. Furthermore, they set the stage for future studies on the role of saprobic fungi in inducing resistance of host plants to pathogens.     

Some factors affecting production of pectic enzymes by Aspergillus niger

The effects of varying cultural conditions were assessed for the production of pectic enzymes in a strain of Aspergillus niger, isolated from decaying orange fruit. Polygalacturonase and pectinmethylesterase were found to be inducible by polygalacturonic acid and pectin in the medium, respectively. Ammonium sulphate was the best nitrogen source for the production of both enzymes. There were variations in enzyme levels produced in culture filtrates with age of the culture, the highest levels being in 4-day-old cultures. The temperature and pH also had marked effects on the production of pectic enzymes with the best conditions being 40°C and pH 5, respectively. Surface culture technique gave appreciable enzyme yield, while agitation had an inhibitory effect on enzyme production.     

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.     

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.     

Biological Control ofMonilinia laxaandFusarium oxysporumf. sp.lycopersiciby a Lytic Enzyme-ProducingPenicillium purpurogenum

Biological control experiments were conducted with the lytic enzyme-producing fungusPenicillium purpurogenumagainst the plant pathogensMonilinia laxaandFusarium oxysporumf. sp.lycopersici.Applications ofP. purpurogenumto peach shoots previously inoculated withM. laxareduced lesion length and extent of pathogen colonization of shoots by 90 and 80% (P ≤ 0.05), respectively, comparable to the level of disease control obtained with the fungicide captan. Disease severity in tomato plants inoculated withF. oxysporumf. sp.lycopersiciwas decreased by 30% (P ≤ 0.05) with the biological treatment. The fungusP. purpurogenumproduced β-1,3-glucanase and chitinase activities in liquid culture that were inducible by cell walls and live mycelium ofM. laxabut not ofF. oxysporumf. sp.lycopersici.Crude filtrates or crude enzyme preparations ofP. purpurogenumcultures with lytic enzyme activities produced lysis of hyphae and spores ofM. laxaandF. oxysporumf. sp.lycopersici.These lytic effects were strong inM. laxaand ended in complete dissolution of mycelium. The induction of lytic enzymes byM. laxaand the effects of lytic enzymes on mycelia of the pathogens in relation to the different degrees of biological control obtained are discussed.     

PECTIC ENZYMES SECRETED BY VERTICILLIUM DAHLIAE AND THEIR ROLE IN THE DEVELOPMENT OF THE WILT DISEASE OF COTTON

An isolate of Verticillium dahliae obtained from Uganda was highly virulent to young cotton plants under greenhouse conditions. A hyaline variant which often appeared in culture was as virulent as the parent isolate, but preliminary experiments indicated that it did not survive as long in unsterile soil. The parent isolate grew rapidly in cotton plants after root inoculation and was isolated from stems and leaves well before the appearance of disease symptoms visible to the naked eye.
Protopectinase was produced in the absence of pectic materials, but more active preparations were obtained when media contained pectic substances. In general, there was a close correspondence between the protopectinase activity of culture filtrates and the toxicity of these filtrates to parenchymatous cells. Some separation of the two activities was obtained by heating enzyme solutions or by plasmolysing the test tissue.
Protopectinase solutions had little pectinesterase activity but rapidly reduced the viscosity of solutions of pectic substances. In general, the properties of protopectinase and the viscosity-reducing enzyme were similar.
Young cotton shoots wilted rapidly when placed in cell-free filtrates from cultures of the pathogen. Wilting was delayed under conditions unfavourable for transpiration. Evidence was obtained which showed that wilting was caused by the uptake of thermostable compounds of high molecular weight which impeded the upward flow of the vascular sap. Pronounced vascular browning was obtained only when solutions containing protopectinase were used. Wilting and vascular browning were obtained with solutions having little pectinesterase activity; in contrast, a solution having high pectinesterase activity produced relatively little vascular browning.     

Further observations on the bacteriology of jute retting

Bacteria capable of retting jute are present in pond water, in the soil of jute fields, and on the surface of jute plants. They probably enter stems over the whole surface through stomata, as well as through cut ends and leaf scars, after immersion for retting. Under laboratory conditions the shortest period of retting, three days, was achieved withBacillus polymyxa at a temperature of 40°. Retting in a tank was appreciably hastened by using the same water for successive batches of jute. Prolonged immersion involves the risk of over-retting, probably because of the action of cellulolytic bacteria. For natural retting, still the most appropriate process in East Pakistan, large clean ponds are thought to provide the most suitable conditions.     

Measurement of amyloglucosidase usingP-nitrophenyl β-maltoside as substrate

Summary An enzyme-linked assay for the measurement of amyloglucosidase in commercial enzyme mixtures and crude culture filtrates is described. A method for the synthesis of the substrate employed,p-nitrophenyl β-D-maltoside, is also described. The substrate is used in the presence of saturating levels of β-glucosidase. With a range ofAspergillus sp. culture filtrates, an excellent correlation was found for values obtained with this assay and a conventional assay employing maltose as substrate with measurement of released glucose.     

Factors involved in multiplication and survival ofEscherichia coli in lake water

The population of a strain ofEscherichia coli that was resistant to nalidixic acid and streptomycin declined rapidly in samples of sterile and nonsterile Cayuga Lake water and reached an undetectable level in nonsterile water at 24 and 72 hours when counted on eosin-methylene blue (EMB) agar and half-strength trypticase soy agar (TSA), respectively. In sterile lake water amended with 10g amino acids per ml or 0.1 M phosphate,E. coli multiplied exponentially for more than 24 hours. The addition ofRhizobium leguminosarum biovarphaseoli to unamended sterile lake water prevented the decline ofE. coli, and its addition to amended sterile lake water preventedE. coli multiplication. The cell density of this strain ofE. coli declined in the first 8 hours after its introduction into an inorganic salts solution, but the bacterium then grew extensively. This increase in abundance was not observed in the presence ofR. phaseoli, andE. coli counts on half-strength TSA remained unchanged between 8 hours and 6 days. When counted on EMB agar, the abundance of the antibiotic-resistant strain ofE. coli and a strain not selected for resistance increased in solutions containing phosphate and amino acids but declined in the presence of high densities ofR. phaseoli. Many of the cells of the antibiotic-resistantE. coli strain failed to grow on antibiotic-amended EMB agar after introduction of the organism into nonsterile or sterile lake water or into an inorganic salts solution containingR. phaseoli, although colonies appeared on TSA. The data suggest thatE. coli cells grown on rich media suffer a shock when introduced into lake water because of low hypotonicity, the indigenous competing flora, or both. This shock is prevented by either phosphate buffer or by amino acids at low concentration. The shocked bacteria formed colonies on half-strength TSA. Depending on environmental conditions, the presence of a second organism either has no effect or results in an increase or decrease inE. coli numbers.     

Thiamine pyrophosphate,a growth factor forTreponema vincentii andTreponema denticola

Growth ofTreponema vincentii N-9 in complex media was stimulated by culture filtrates ofT. minutum. Thiamine pyrophosphate (TPP) in nanogram quantities could substitute for the stimulatory factor inT. minutum culture filtrates. Characterization of the filtrates indicated that the stimulatory factor was similar to TPP in heat stability and ultrafiltration properties. The oral treponemesT. vincentii andT. denticola required TPP for maximum growth, whereas the genital treponemesT. minutum, T. phagedenis, andT. refringens do not require TPP. The presence of TPP activity in culture filtrates of the genital treponemes was confirmed by using a strain ofNeisseria gonorrhoeae auxotrophic for TPP. TPP activity was not found in culture filtrates of oral treponemes. Culture filtrates of 13 of 14 species representing eight genera found in the microbial flora of the oral cavity also contained TPP activity.     

Additional studies of histoplasmin formation

Summary Culture filtrates of 20 strains ofHistoplasma capsulatum were studied to determine the effect of certain growth conditions on histoplasmin formation. The presence of histoplasmin was denoted by an antigenic titer of 1:4 or higher with the complement fixation test.The data indicated that, in addition to verifying that the strain used affected histoplasmin formation, the morphological condition of the inoculum was extremely important. It was found that most strains which converted readily to the yeast phase at 37° C produced histoplasmin poorly. Tests with different volumes of media also showed that 500 ml volumes of culture media produced histoplasmin with higher titers than 3 liter volumes when cultured at 25° C for six months.Some additional histoplasmin could be liberated by sonification of the mycelial pad from culture filtrates which contained histoplasmin. A few strains produced high titer histoplasmin by the shake method if incubated for three months, but they had low titers after only six weeks.Complement fixation tests with sera from proven cases of histoplasmosis indicated that histoplasmin from a single strain ofH. capsulatum can give identical results with those obtained with histoplasmin from a pool ofH. capsulatum strains if H and M antigen components are present.