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.     

Cellulase and ({beta}-Glucosidase Components in Culture Filtrates from Botryodiplodia theobromae Pat.

Cellulase and ß-glucosidase components in culturefiltrates from Botryodiplodia theobromaePat. were separatedby polyacrylamide-gel electrophoresis and gel-filtration onSephadex. A correlation between the electrophoretic mobilitiesand gel-filtration behaviour of the components was established.Four cellulase components (Ca, Cb, Cc, and Cd) were recognized with approximate molecular weights of 162 000 (Ca), 15 000(Cb), 10 000 (Cc), and 4500 (Cd). Four ß-glucosidasecomponents (Ba, Bb, Bc, and Bd) were also recognized with molecularweights of 112 000 (Ba), 56 000 (Bb), 27 000 (Be), and 13 300(Bd). The cellulase components appeared to be composed of aggregatesof similar subunits. Similarly, the ß-glucosidasecomponents appeared to be aggregates of similar subunits. Similarly,the ß-glucosidase components appeared to be aggregratesof a subunit that differed from the cellulase subunit. The activitiesof the cellulase components differed on different cellulosicsubstrates. Cotton flock was readily solubilized by a mixtureof cellulase components Ca, Cb, and Cc, an effect which wasenhanced in the presence of component Cd. Any appreciable solubilizationof native cotton fibres required component Ca or, more effectively,Cd.     

The cellulolytic enzymes of Botryodiplodia theobromae Pat. Separation and characterization of cellulases and β-glucosidases

1. Filtrates from cultures of different ages of Botryodiplodia theobromae Pat. were fractionated by gel filtration, ion-exchange chromatography and polyacrylamide-gel electrophoresis. 2. Five cellulases (C1, C2, C3, C4 and C5) were found, and their molecular weights, estimated by gel filtration, were 46000–48000 (C1), 30000–35000 (C2), 15000–18000 (C3), 10000–11000 (C4) and 4800–5500 (C5). 3. Cellulase C5 was absent from old culture filtrates. 4. Cellulase C1 had little or no activity on CM-cellulose (viscometric assay), but degraded cotton flock and Whatman cellulose powder to give cellobiose only. 5. The other components (C2–C5) produced cellobiose and smaller amounts of glucose and cellotriose from cellulosic substrates and were more active in lowering the viscosity of CM-cellulose. 6. The ratio of activities assayed by viscometry and by the release of reducing sugars from CM-cellulose increased with decrease in the molecular weights of cellulases C2–C5. 7. Cellobiose inhibited the activities of the cellulases, but glucose stimulated at low concentrations although it inhibited at high concentrations. 8. A high-molecular-weight β-glucosidase (component B1, mol.wt. 350000–380000) predominated in filtrates from young cultures, but a low-molecular-weight enzyme (B4, mol.wt. 45000–47000) predominated in older filtrates. 9. Intermediate molecular species of β-glucosidase (B2, mol.wt. 170000–180000; B3, mol.wt. 83000–87000) were also found. 10. Cellulases C2–C5 acted in synergism with C1, particularly in the presence of β-glucosidase.     

Periodicity of Wood Formation in Some Trees of Lowland Rainforest in Nigeria

Freshly prepared chlor-zinc-iodide was used to determine theperiodicity of wood formation at breast height (144 cm fromthe ground) in the trunks of some trees growing in the LowlandRainforest around Ibadan, Nigeria. Wood formation shows seasonal periodicity in the plants studied.The cambial derivates on the xylem side differentiate into woodcells, which at a certain stage of differentiation have abundantcellulose in their secondary walls. The cellulose stains deepblue in chlor-zinc-iodide. This has been used as a criterionfor deciding that wood formation has started. When no cellswith deep blue staining secondary walls are found the cambiumis known to be dormant or quiscent. The resumption of cambial activity is correlated positivelywith bud break and unfolding of new leaves. In Bombax buonopozenseP. Beauv. the relationship may be obscured by local cambialactivity induced by injury. Wood formation stops in the trunks either towards the end ofthe rainy season or at the early part of the dry season. Itstarts either during the dry season or at the beginning of therainy season; but the bulk of the wood is formed during therainy season. Cambial activity stops in most cases before leaffall. At the cessation of wood formation the fully lignifiedxylem elements abut on the xylem mother cells or on xylem cellswith incompletely thickened cell walls. Presence or absence of a starch-free-zone and the noding ofthe vascular rays also give indications of seasonal periodicityin wood formation. Growth rings are periodic and one growthring is generally formed each year.     

Production of Cellulolytic Enzymes by Botryodiplodia theobromae, Pat.

Carboxymethylcellulase (CM-cellulase) and ß-glucosidaseactivities were induced in cultures of Botryodiplodia theobromae,Pat. Both growth of the fungus and CM-cellulase production werebetter with sodium nitrate as nitrogen source than with eitherammonium nitrate or ammonium chloride. Growth, per unit nitrogensupplied, was greater with glutamic acid and aspartic acid asnitrogen sources than with sodium nitrate; this was probablybecause the fungus utilized the carbon of these amino-acids.Most ß-glucosidase activity was formed with ammoniumchloride or ammonium nitrate as nitrogen source. Glucose inhibited the activity of ß-glucosidase markedly.Low glucose concentration (c. 0.003 M) stimulated CM-cellulaseactivity but concentrations above 0.05 M inhibited. The formationof both enzymic activities was repressed in the presence ofglucose. ß-glucosidase activity was more thermolabilethan that of CM-cellulase.     

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.     

The extracellular enzymes of Coniophora cerebella

1. The extracellular enzymes present in the culture filtrates of Coniophora cerebella grown on various carbohydrate carbon sources were investigated. Enzymes that degraded cellulose derivatives, hemicellulose fractions, starch, laminarin, pectin and several oligosaccharides were detected. 2. All the polysaccharide-degrading activities were adaptive except for that acting on laminarin. 3. The culture filtrates degraded native cellulose to only a very limited extent. 4. The hemicellulase activity included enzymes acting on all the major components of wood hemicellulose. 5. The main starch-degrading enzyme was a glucoamylase. 6. Laminarin-degrading activity was produced when cellulose, hemicellulose or starch was used as carbon source for the fungus and it may be involved in the re-utilization of hyphal carbon or of a reserve polysaccharide synthesized during active growth of the organism.     

(+)-7-Iso-jasmonic acid and related compounds from botryodiplodia theobromae

Metabolites produced by Botryodiplodia theobromae (synonym Lasiodiplodia theobromae) possessing plant growth regulating activities were shown to be the 7-epimer of jasmonic acid and its derivatives.     

Quantum Flux Density as a Factor Controlling the Rate of Growth, Carbohydrate Partitioning and Wood Structure of Betula pubescens Seedlings

The ability of Betula pubescens seedlings to acclimate to arange of quantum flux densities is examined, paying particularattention to the quantity, rate and type of wood produced. Betulapubescens seedlings were germinated and grown for 2 months ata quantum flux density of 440 µmol m^–2 s^–1,then transferred to 440, 244 or 89 µmol m^–2 s^–1. Seedlings transferred to reduced fluence rates were able tosurvive and grow. Acclimation was associated with an increasedallocation of carbon to stem elongation and leaf-area production,this occurred at the expense of root storage carbohydrate androot growth. The rate of wood production was shown to be directlyrelated to quantum flux density. Stem wood structure at smallquantum flux density showed an increase in proportion of fibres,while root wood structure in this regime showed a greater proportionof vessels. In general the activity of phenylalanine ammonia-lyase(PAL) showed little significant difference between treatments.The implications of these changes in wood structure are discussedand related to wood production and PAL activity. Betula pubescens Ehrh., birch., shade tolerance, carbohydrate partitioning, relative growth rate, wood structure, phenylalanine-ammonia-lyase     

Regulation of Storage Protein Synthesis by Sulphur and Nitrogen Nutrition in Cultured Immature Caryopses of Barley

Techniques are described for the culture of developing barley(Hordeum vulgare L.) caryopses. Over a 7 d period in culturethe dry weights and the amounts of starch and protein increasedby at least twofold. Growth was sustained for at least 20 d.The effects of glutamine and cysteine on the amount and compositionof the hordein storage proteins were also studied. Glutaminestimulated total hordein accumulation but caused a disproportionateincrease in the amount of the S-poor ‘C’ hordeinswhen supplied at 100 mol m^–3. Addition of cysteine at1·0 mol m^–3 did not increase the amount of Srich‘B’ hordeins. The results suggest that althoughisolated caryopses are able to take up sucrose and glutamineand convert them to starch and protein there is some limitationin their ability to convert externally supplied cysteine intoproteins. Key words: Hordeum vulgare L., Caryopses, Glutamine, Cysteine, Storage proteins     

Are bacteria an important food source for rotifers in eutrophic lakes?

In situ grazing measurements using fluorescent particles of0.5, 2.4 and 6.3 µm diameter in eutrophic Lake Loosdrecht(The Netherlands) showed that Anuraeopsis fissa, a small rotifer,filtered the smallest, bacteria-sized particles as efficientlyor more efficiently than the larger particles. In contrast,three other rotifer species (Brachionus angularis, Filinia longisetaand Pompholyx sulcata) filtered the bacteria-sized particlesless efficiently than the larger particles. Both Keratella cochlearisand Conochilus unicornis only ingested the bacteria-sized particles.Anuraeopsis fissa had a higher uptake of fluorescent bacteria-sizedparticles than K.cochlearis, both in 1 µm filtrate oflake water and in lake water. Within both species, uptake didnot differ between juveniles and adults. When cultured on threedifferent size fractions of lake water (1, 3 and 15 µmfiltrate) in July, all rotifer species declined in numbers onthe 1 and 3 µm filtrates, while A.fissa and B.angularisincreased in numbers on the 15 µm filtrate. The high abundanceof small bacteria in the lake water could not support rotiferpopulations. It is concluded that bacteria are not a suitablefood source of high quality for A.fissa because its populationdoes not grow even though the bacterial concentration was higherthan its estimated threshold food concentration. In August,when individually cultured, the mortality was high for all species,but especially for F.longiseta. The lifespan of K.cochleariswas reduced in the 1 and 3 µm filtrates of lake water,compared with in the 15 µm filtrate. The lifespan of A.fissawas similar in all filtrates, but reproduction was reduced inthe 1 and 3 µm filtrates, as in Keratella. On the 15 µmfiltrate, their ages at first reproduction and growth ratesdid not differ. Individuals of A.fissa older than 4 days showeda higher survival in the 15 µm filtrate than in the othertwo filtrates, as did K.cochlearis throughout its life. Hence,bacteria seem to be a more important food source for youngerindividuals of A.fissa than of K.cochlearis.     

Increase in cellulase activity in cultured soybean cells caused by Rhizobium japonicum

Cell envelopes of Rhizobium increased the cellulase activityof soybean (Glycine max cv. Kingen No. 1) cells in a suspensionculture within one hour. Cellulase activity was assayed by measuringthe decrease in viscosity of a carboxymethyl cellulose solution.The effective strains for soybean caused a larger increase incellulase activity (1.4–4.1 times the control level) thanthe ineffective strains (0.8–1.6 times the control level).The cell envelopes of effective strains also caused rupturesof some soybean cells. (Received December 21, 1979; )     

Efficacy of new EC formulation derived from garlic creeper (<Emphasis Type="Italic">Adenocalymma</Emphasis><Emphasis Type="Italic">alliaceum</Emphasis> Miers.) against anthracnose and stem end rot diseases of mango

Different leaf extracts of Garlic creeper (Adenocalymma alliaceum Miers.) using water and solvents were prepared and they were screened for their antifungal activity against Colletotricum gloeosporioides Penz. and Botryodiplodia theobromae Pat. causal agents of mango post harvest diseases viz., anthracnose and stem end rot respectively. Among the extracts tested, chloroform extract was found to be highly effective in inhibiting the spore germination of C. gloeosporioides by 84.62% and B. theobromae by 84.50% followed by methanol extract. The extracts were also inhibitory to the mycelium. Ammonium sulphate precipitation and SDS–PAGE analysis of the extract indicated the presence of a major protein with a molecular weight at 65 kDa. Two blue spots at Rf 0.96 and 0.80 was also observed in TLC analysis and the presence of tannic acid, resorcinol was evident from HPLC analysis. Treatment of fruits with leaf extract of A. alliaceum increased the activities of peroxidase, polyphenol oxidase, phenylalanine ammonia-lyase and sugars with significant reduction in starch, phenolics, protein and ascorbic acid. The extract was partially purified and formulated as ADENOCAL 60 EC for the management of post harvest diseases of mango fruits.     

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.     

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.     

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.     

Further Observations on Fungi Inhabiting Pine Stumps

Growth-rates, both on malt agar and in stump wood, of earlystump colonizers such as Fomes annosus, Pemophora gigantea,Stereum sanguinolentum, and Leptographium lundbergii are higherthan those of later-colonizing agarics. The initial invasionof stump roots usually proceeds via the stump body by fungiwhich infect the cut surface. Although stumps may dry out appreciably shortly after felling,in later stages of decay the moisture content of the wood oftenincreases considerably. Stump decomposition is associated witha succession of decay fungi, the ‘Peniophora’, ‘Hypholoma’,and ‘Tricholoma’ stages being distinguished. Severalphycomycetes and fungi imperfecti colonize decayed, but notfresh, stump wood. P. gigantea is shown to be a vigorous competitor of F. annosusfor initial colonization of stumps: it may also replace F.annosus in stumps. Gliocladium viride and Trichoderma viridecompete with wood-rotting basidiomycetes and sometimes replacethem in very decayed wood.     

Cortical Bundles in the Persistent, Photosynthetic Stems of Cacti

We examined 62 species in 45 genera of the cactus subfamilyCactoideae; all had collateral cortical bundles that permeatedthe broad, water-storing inner cortex and extended to the baseof the outer, photosynthetic palisade cortex. Mean distancebetween cortical bundles was 0.75 mm, similar to the mean spacing(0.74 mm) of veins in leaves of Pereskia, a genus of relictleaf-bearing cacti. In 16 species, both young and extremelyold stem cortex was available for study: in all of these, olderbundles had larger amounts of phloem than did younger bundles,indicating that phloem had been produced for many years. Inten species, older bundles also had more xylem than youngerbundles. In two genera (Rhipsalis and Selenicereus) there werecaps of primary phloem fibres, and in a single species (Pilosocereusmortensenii) cortical bundle xylem contained libriform fibres.All cortical bundle tracheary elements were narrow (radius range,0.91–8.2 µm; mode, 1.8–2.7 µm), similarto Pereskia leaf vein elements (radius range, 1.8–2.7µm); this was much narrower than stem wood vessels (radiusrange, 10–42 um; mode, 23–28 µm). Longitudinalconduction of water and nutrients probably occurs predominantlyin stem wood, with cortical bundles maintaining the broad, voluminouscortex, the outer part of which is the plant's photosynthetictissue and the inner part of which stores water and starch.The cortex of the Cactordeae contains numerous leaflike characters;homeotic genes may be involved in its morphogenesis. Cactaceae, cortical bundles, homeotic, xylem, phloem, evolution     

Cyclodextrins are likely to induce cyclodextrin glycosyltransferase production inBacillus macerans

Cyclodextrin glycosyltransferase (CGTase) activity was monitored inBacillus macerans culture fluids up to 56 h incubation time using glucose (G₁), maltose (G₂), maltotriose (G₃), maltoheptaose (G₇), α-,β-,γ-cyclodextrins (CDs) and soluble starch as carbon sources. Highest maximum specific growth rates (μ_max) were observed with glucose, γ-CD and soluble starch (μ_max values were 0.86, 0.74 and 0.69/h, respectively) while the maximum viable cell numbers were always within the range of 2.3–7.1×10¹¹ CFU/mL independently of the carbon source used. Highest CGTase production was found in the presence of soluble starch and G₇ (55.0 and 35.4 nkat/mL, respectively), these saccharides being easily transformed to CDs by CGTase. Moreover, when culture media were supplemented with cyclic malto-oligosaccharides the CGTase activities were about twice higher (19.6–20.6 nkat/mL) than those obtained with the linear G₂ and G₃ saccharides (8.9 and 11.3 nkat/mL, respectively) which give rise only to negligible quantities of CDs. CDs, which are the major end products of the action of CGTase, are regarded thus as the likely physiological inducers of the enzyme.     

Mutant genes at the r and rb loci affect the structure and physico-chemical properties of pea seed starches

Mutant genes at two loci, r and rb, known to encode genes affectingthe starch biosynthetic pathway, were studied for their effecton the structure and gelatinization of pea seed starches. Comparisonswere made using starches from four lines {RRRbRb, rrRbRb, RRrbrb,and rrrbrb), near-isogenic except for genes at these two loci.All the starches had C-type X-ray diffraction patterns, butdifferent contents of ‘A’ and ‘B’ polymorphs.The presence of a mutation at either locus increased the ‘B’polymorph content in the starches, although the influence ofthe r mutation was much greater than that of rb. Differenceswere discovered in the crystalline stucture of the rrRbRb starchwhich correlated with a high content of amorphous phase as wellas with the changes in amylopectin structure. In addition, changesin the crystalline structure of this sample correlated witha lack of co-operative transition during starch gelatinizationin excess water. The RRrbrb starch had a greatly increased enthalpyof gelatinization in excess water compared with the wild-typestarch. It is proposed that this effect is connected with specificcharge interactions between the molecules in the starch granule.The rrrbrb starch had parameters of crystalline structure andgelatinization which reflected the different influences of thetwo genes. With regard to gelatinization, this starch had relativelywide co-operative transition and low enthalpy and a very highpeak temperature of transition. Key words: Pisum sativum, starch structure, genetic effects, rugosus mutants