Cellulolytic enzymes associated with the fruit rots of Citrus sinensis caused by Aspergillus aculeatus and Botryodiplodia theobromae

Botryodiplodia theobromae and Aspergillus aculeatus were inoculated on carboxymethylcellulose (CMC) medium and filter papers. The hydrolysis of the CMC medium and degradation of the filter papers were observed, indicating the production of the C_l and C_x cellulases by the two rot pathogens. The C_l and C_x enzymes were also detected in filtrates of rotted orange fruits incited by the two rot pathogens.The cellulases could not induce rot development on their own. However, when they were added to pectinases in an enzyme inoculum, the incubation period for inducing rot development was shorter; thus establishing a secondary role for the cellulases in the rot development. This secondary role of the cellulases produced by the two fungi was found to be at peak at pH 7 and a temperature range of 25°–30 °C in the two fungi.     

Minor root rot pathogens of cassava (Manihot esculenta Crantz) in Nigeria

Many different species of fungi are often isolated from rotted cassava root tubers and pathogenicity studies have often implicated Botryodiplodia theobromae and Fusarium solani as the major causal pathogens. Consequently, more attention has often been focused on Botryodiplodia theobromae and Fusarium solani with little or no attention on the other minor pathogens. Considering the increasing importance of cassava to the Nigerian economy and the fact that minor root rot pathogens of cassava today could become major tomorrow, the aim of this research is to determine the incidence, pathogenicity and symptoms of the minor root rot pathogens in cassava from cassava fields within the derived savanna and the humid forest of Nigeria. Isolation of associated fungi was done on rotted root samples and the pathogenicity of these isolates were established by inoculating them into healthy cassava tuberous roots and subsequently reisolating them from resulting rotted tissue. The less frequently isolated fungi where Macrophomina sp., Trichoderma sp., Aspergillus niger, Aspergillus flavus, Sclerotium rolfsii and Fungus ‘A’ (a yet to be identified fungus). Repeated experiments confirmed a constant relationship between inoculated fungus and the resulting rotted tissue colour. The root rot tissue colours associated with inoculated pathogens in the laboratory were identical with the pathogens colony colour on potato dextrose agar.     

The Adsorption of a Polygalacturonase Isoenzyme of Botryodiplodia theobromae on Plant Tissues and the Implication on the Pathogenicity of the Fungus

The polygalacturonase isoeazyme (PG 3) of Botryodiplodia theobromae extracted from rotted sweet potato was adsorbed by sweet potato, potato, carrot, bean stem and tomato fruit to various degrees. Adsorption was greater with sweet potato and tomato fruit tissues. Carrots, bean stem and potato absorbed the enzyme to more or less the same degree. The enzyme was not adsorbed on tomato stem. A spore/mycelial suspension of B. theobromae infected the test tissues to various degrees. The enzyme completely macerated sweet potato roots, potato tubers and tomato fruits within 5 h while the bean stem and onion tissues were little affected by the enzyme. The tomato stem was neither infected by the fungus nor macerated by the enzyme.     

Cellulolytic Activities of Botryodiplodia theobromae Pat

Botryodiplodia theobromae Pat., a ‘stain’ fungusisolated from stained and decaying wood of Bombax buonopozenseP. Beauv., attacked isolated blocks of Bombax wood. In theseit perforated the cell walls and formed elongated cavities withpointed ends, usually in chains, in the secondary wall. Thefungus attacked cellulose in a similar manner to soft rot fungi.It used starch and other saccharides present in the wood ofBombax buonopozense as initial substrates before degrading thecellulose and hemicellulose components of the wood. It did notdegrade the lignin component of the wood. Cellulase, ß-glucosidase, and amylase activities weredetected in culture filtrates of Botryodiplodia theobromae.Cellulase synthesis was delayed when the wood powder in themedium contained starch and saccharides. Amylase, too, behavedlike an inducible enzyme and disappeared from the cultures onexhaustion of starch. Electron microscopy showed that cellulose extracted from thewood of Bombax buonopozense was fragmented into small particleson treatment with the culture filtrates of Botryodiplodia theobromae.Cellobiose, glucose, and another saccharide, probably cellotriose,were identified as the products of enzymatic hydrolysis of cellulose. These results are discussed in relation to the attack and degradationof the wood of Bombax buonopozense by Botryodiplodia theobromaein nature.     

Studies on the latent infection of Botryodiplodia theobromae Pat. on Citrus sinensis (Li.) Osbeck fruit

The latent infection caused by Botryodiplodia theobromae on Citrus sinensis fruit was investigated. Isolations from washings from mature green fruit surfaces showed the occurrence of B. theobromae, Rhizopus stolonifer and Trichoderma sp. The histological studies on fruit tissues revealed some structural and physiological changes between ripe and unripe fruits while investigations on the host-parasite relations could not detect any propagule of B. theobromae at the early stage of infection. The effects of rind extracts obtained at 60°, 80° and 100°C from mature green, half ripe and full ripe fruits when cooled were tested on the growth of three fruit moulds. Extracts from healthy mature green fruit rind obtained at 80°C showed significant inhibition of growth of B. theobromae while R. stolonifer and C. paradoxa could not grow in this medium.     

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.     

Storage losses of taro corms in the British Solomon Islands Protectorate

In two storage trials, corms of taro (Colocasia esculenta) decayed and became unfit for human consumption after 1 to 2 weeks. There were three fairly distinct types of rot: A dry rot caused by Fusarium solani, a spongy black rot caused by Botryodiplodia theobromae, and a Sclerotium rot caused by S. rolfsii. Although it was found that, under humid conditions, all three fungi could penetrate and rot undamaged corms most natural infection is thought to occur through wounds. None of the pathogens rotted corms at low humidity.     

Effect of fungi–termite interaction on the chlorophyll content of three rice varieties grown on ultisol soil of Ikenne,south-west Nigeria

The effect of fungi–termite interaction on three rice varieties was conducted in a screen house at the Africa Rice Center (AfricaRice) Ibadan, Nigeria. Of the 10 fungi species (Fusarium verticilloides, Trichoderma sp., Aspergillus niger, Aspergillus flavus, Macrophoma sp., Neurospora sp., Botryodiplodia theobromae, Penicillum sp., Rhizopus sp. and Sclerotium rolfsii) isolated from termites, soil and rice plants, F. verticilloides, Trichoderma sp. and B. theobromae were used for the interaction study. Each fungus was inoculated singly and in combinations with termite into the root of each rice variety in potted soil. Leaf samples were taken to measure the chlorophyll content which is a major parameter to estimate effect of termite–fungi interaction. The chlorophyll content of the inoculated rice plants was significantly reduced when compared with the control. Fungi interaction with termite had significant reduction on the chlorophyll content. The synergistic relationship between the fungi and the termite was discussed.     

Cellulolytic enzymes associated with the fruit rots of Citrus sinensis caused by Aspergillus aculeatus and Botryodiplodia theobromae

Botryodiplodia theobromae and Aspergillus aculeatus were inoculated in carboxymethylcellulose (CMC) medium and on filter papers. Hydrolysis of the CMC medium and degradation of the filter papers were observed, indicating the production of C1 and Cx cellulases by the two rot pathogens. The C1 and Cx enzymes were also detected in filtrates of rotted orange fruits obtained by infection with the two pathogens. The cellulases could not induce rot development on their own. However, when they were added to pectinases in an enzyme inoculum, the incubation period for inducing rot development was shorter, thus establishing a secondary role for the cellulases in the rot development. Optimum conditions for the action of the cellulases included a neutral pH and temperature ranging from 25 to 30 degrees C.     

Some extracellular enzymes associated with two tomato fruit spoilage molds

The production of amylolytic, cellulolytic and pectinolytic enzymes by Aspergillus flavus and A. fumigatus was investigated. The two fungi were cultured on wheat offal and liquid crystalline carboxymethylcellulose media. A. flavus produced amylases on basal and starch containing media while A. fumigatus could only produce amylases on starch medium. The cellulolytic activities of filtrates from culture or infected fruits showed that A. flavus produced lesser quantities of cellulolytic enzymes than A. fumigatus. At 25 °C and at a pH range of 6–8, A. flavus best produces amylases and cellulases, while A. fumigatus showed highest activities of the two enzymes at 35–40 °C and at pH 7.0. Two pectinolytic enzymes — polymethylgalacturonase and pectinmethyltrans-eliminase — were identified in vivo with the two molds. An endopolygalacturonase in addition to these two pectinolytic enzymes was well associated with A. fumigatus.     

Root-surface mycoflora of cassava (Manihot esculenta) and post harvest rot of the tubers

The root-surface mycoflora of cassava were isolated from roots washed in serial changes of sterile distilled water and plated out on potato-dextrose agar. A small group of fungi which included Aspergillus niger, Botryodiplodia theobromae, Fusarium solani, Penicillium javanicum, Penicillium sp., and Trichoderma sp. were found to be consistently associated with the root surface. While the isolates, B. theobromae and F. solani were found to be aggressively pathogenic on freshly harvested cassava tubers causing extensive rot, A. niger was only mildly so. The root-surface mycoflora, therefore, includes fungi which have been reported as the most important in postharvest deterioration of the tubers. The removal of the rhizoplane microflora by surface-sterilization using calcium hypochlorite or Clorox and subsequent incubation in loosely tied polyethylene bags extended the storage life of the tubers considerably.     

Pectolytic enzymes produced byRhizoctonia bataticola in culture andRhizoctonia-infected tissues of potato tubers (Solanum tuberosum L.)

Rhizoctonia bataticola produced both polygalcturonase (PG) and pectin-methyl-esterase (PME) in vitro and in vivo, the former releasing more reducing materials in reaction mixtures. Extracts of healthy tissues ofSolanum tuberosum contained PME but no PG. Salt extraction of healthy and diseased tissues increased the amount of PME obtained. The activity of PME produced in vivo increased after dialysis and addition of salt to reaction mixtures whereas the PME produced in vitro was neither affected by dialysis nor influenced by addition of salt to the preparations. Enzyme filtrates from culture andRhizoctonia-infected tissues induced rot symptoms onto sterilized pealed healthy tubers ofS. tuberosum whereas the extracts from healthy tissues did not.     

Studies on the pectolytic enzymes ofaspergillus terreus Thom

Summary Aspergillus terreus, the causal organism of fruit-rot of apples produced pectin methylesterase (PME) and depolymerase (DP) on a number of substrates. The production of polygalacturonase (PG) was restricted to few substances only and appeared to be adaptive. PME as well as hydrolytic activities were maximum when cultures were incubated at 25°C. The pH of the substrate for maximum PME and hydrolytic activities was 5.5 and 5.0 respectively.     

The role of micro-organisms in the decay of coconut leaf thatch

Five fungi associated with the decay of coconut leaf thatch, a low cost organic roofing material used widely in Kerala (India), were isolated and identified as Aspergillus niger, A. tamarii, Penicillium citrinum, Pestalotiopsis palmarum and Botryodiplodia theobromae. Inoculation studies carried out in a controlled humidity chamber providing optimum conditions for the growth of fungi followed by measurement of tensile strength indicated that the species actively involved in reducing the tensile strength of the leaf and thus shortening the life span of the leaf are A. niger and P. citrinum. The other three fungi are possibly involved only in causing surface damage to the leaf.     

Oxalic acid production by Fusarium oxysporum Schlecht and Botryodiplodia theobromae Pat., post-harvest fungal pathogens of yams (Dioscorea rotundata L.) and detoxification by Bacillus subtilis CM1 isolated from culturable cowdung microflora

Abstract

Fusarium oxysporum Schlecht and Botryodiplodia theobromae Pat., two important post-harvest pathogens of yam (Dioscorea rotundata L.) tubers in storage were found to produce oxalic acid (OA) in vitro and in vivo. The rate of OA accumulation was proportional to fungal growth (cell mass) in Potato Dextrose liquid medium during 10 days incubation period. Further, simultaneous co-culturing of either of the fungi with Bacillus subtilis CM1 isolated from cowdung culturable microflora resulted in 92% reduction in OA accumulation compared with that in the culture of the individual fungus. The effect was more prominent in pH 5 – 6 than in pH 7 – 8. B. subtilis CM1 was capable of detoxifying OA and several proteins were detected in the culture filtrates when it was grown in peptone-mineral salt medium containing OA. SDS-PAGE analysis of 70% ammonium sulphate fraction of the culture filtrate exhibited the presence of a predominant 97 kDa protein.     

Bacterial and fungal metabolites of rhizospheric microflora of cotton antagonistic to Rhizoctonia solani Kühn

Summary Growth response ofRhizoctonia solani Kühn, the damping-off fungus, to metabolites of selected antagonistic rhizospheric bacteria and fungi of some Egyptian cotton varieties, namely, two strains ofBacillus subtilis Cohn,Aspergillus terreus Thom, andAspergillus flavus Link produced in culture media containing nitrate- or ammonium-nitrogen sources, proved the potency ofB. subtilis metabolites in inhibitingR. solani mycelial growth whether from nitrate- or ammonium-nitrogen culture media. Metabolite filtrates ofB. subtilis (II) are more potent than those ofB. subtilis (I). Increasing concentration of bacterial metabolite filtrates resulted in a decreased mycelial dry weight ofR. solani. The bacterial inhibitory factor forR. solani mycelial growth is partially affected by heat. Metabolite filtrates ofA. terreus from nitrate-nitrogen are slightly more potent than from ammonium-nitrogen culture media while an opposite relation is evident withA. flavus metabolites. Growth responses ofR. solani to different experimental dilutions of metabolite filtrates ofA. terreus andA. flavus proved the intervention of the nutritive factor in witholding growth of the damping-off fungus.     

Studies on inhibitory activities of antibodies against phytopathogenic fungi: an immunological approach

The major problem in agriculture is that of the fungal pathogens. In this era, where biological control is at focus, and is the centre of crop protection as well as environmental protection, the synthesis of new bio bodies is of utmost need. Fungicides available in the market, though of potential, are pathogen specific and highly pollutive. An attempt was made to raise polyclonal antibodies against Aspergillus niger. Following the particular standardised immunisation schedule, regular injections and periodic tapping were carried out. The IgG purified was used to check cross-reactivity with different crop fungi like Fusarium solani, Fusarium oxysporum f. sp. cicer (FOC), Rhizoctonia bataticola, Botryodiplodia theobromae, Curvularia sp., Alternaria porri and also with Aspergillus niger by two different methods. Liquid test media and the radial growth inhibition test performed in solid media were used to check the inhibition of fungi and cross-reactivity. The results were subjected to analysis of variance (ANOVA) by using Tukey's test at the significance level of p < 0.05. The antibodies were active against all the fungi for more than 15 days except for A. niger in which from the seventh day onwards spore germination was observed. The probable role of antibodies to detect the common antigenic molecule that may be present in all the tested fungi and their role in inhibiting these fungi is discussed.     

Disease-induced changes in carotenoid content of edible yams (Dioscorea spp.) infected by Botryodiplodia theobromae and Aspergillus niger

When pieces of Dioscorea dumentorum, Dioscorea alata and Dioscorea cayenensis were inoculated with suspensions of Botryodiplodia theobromae and Aspergillus niger and incubated at room temperature, the quantities of carotenes and xanthophylls were decreased by infection. The quantities of carotenes and xanthophylls were highest in D. dumentorum followed by D. cayenensis; D. alata had the lowest amounts.     

Toxicity of citric and succinic acids for the pycnidiospores ofBotryodiplodia theobromae

The toxic effect of citric and succinic acids on the germination of the pycnidiospores ofBotryodiplodia theobromae, mycelial growth and the killing rate of theB. theobromae spores was investigated. The percentage inhibition of germination of viable fungal spores by 0.01% succinic or citric acid ranged between 51.6 and 58.1%, respectively.B. theobromae was found to grow in 2% malt extract broth at 28°C at the rate of 0.13 CFU/h. Citric acid exhibited a higher killing rate of 0.26 CFU/h and was more effective against the germination of the fungal spores. At concentrations of 0.3% and above, citric acid could be used as pre- and post-infectional fungicide.     

The antibiotic effect of culture filtrates of some soil fungi on rhizobial growth in cultures

Summary Four strains ofRhizobium sp. from peanut (Arachis hypogaea L.) root nodules were tested for their sensitivity to metabolites (culture filtrates) of more than ten common soil inhabiting fungi, in yeast extract mannitol agar and broth cultures. Among the rhizobial strains tested strain CB-530, BU-1 and BU-2 were not sensitive to metabolites ofMyrothecium roridum andFusarium moniliforme whereas CB-1024 was sensitive. Culture filtrates ofGliocladium roseum, Thielavia basicola andDrechslera pedicellata inhibited the strains CB-530 and BU-2 but not CB-1024. Most of the soil inhabiting fungi tested were inhibitory to rhizobial growthin vitro and very few were stimulatory, their effect in either case being strain specific.