Broadscale Specificity in a Bark Beetle–Fungal Symbiosis: a Spatio-temporal Analysis of the Mycangial Fungi of the Western Pine Beetle

Whether and how mutualisms are maintained through ecological and evolutionary time is a seldom studied aspect of bark beetle–fungal symbioses. All bark beetles are associated with fungi and some species have evolved structures for transporting their symbiotic partners. However, the fungal assemblages and specificity in these symbioses are not well known. To determine the distribution of fungi associated with the mycangia of the western pine beetle (Dendroctonus brevicomis), we collected beetles from across the insect’s geographic range including multiple genetically distinct populations. Two fungi, Entomocorticium sp. B and Ceratocystiopsis brevicomi, were isolated from the mycangia of beetles from all locations. Repeated sampling at two sites in Montana found that Entomocorticium sp. B was the most prevalent fungus throughout the beetle’s flight season, and that females carrying that fungus were on average larger than females carrying C. brevicomi. We present evidence that throughout the flight season, over broad geographic distances, and among genetically distinct populations of beetle, the western pine beetle is associated with the same two species of fungi. In addition, we provide evidence that one fungal species is associated with larger adult beetles and therefore might provide greater benefit during beetle development. The importance and maintenance of this bark beetle–fungus interaction is discussed.     

Interactions Between the Yeast <Emphasis Type="Italic">Ogataea pini</Emphasis> and Filamentous Fungi Associated with the Western Pine Beetle

Ecologically important microbes other than filamentous fungi can be housed within the fungal-transport structures (mycangia) of Dendroctonus bark beetles. The yeast Ogataea pini (Saccharomycetales: Saccharomycetaceae) was isolated from the mycangia of western pine beetle (Dendroctonus brevicomis) populations in northern Arizona (USA) with a frequency of 56%. We performed a series of in vitro assays to test whether volatile organic compounds produced by O. pini affected radial growth rates of mutualistic and antagonistic species of filamentous fungi that are commonly found in association with the beetle including Entomocorticium sp. B, Ophiostoma minus, Beauvaria bassiana, and an Aspergillus sp. We determined the compounds O. pini produced when grown on 2% malt extract agar using a gas chromatography/mass spectrometry (GC/MS) analysis of headspace volatiles. Volatiles produced by O. pini on artificial media significantly enhanced the growth of the mutualistic Entomocorticium sp. B, and inhibited growth of the entomopathogenic fungus B. bassiana. GC/MS revealed that O. pini produced ethanol, carbon disulfide (CS₂), and Δ-3-carene in headspace. The results of these studies implicate O. pini as an important component in D. brevicomis community ecology, and we introduce multiple hypotheses for future tests of the effects of yeasts in the symbiont assemblages associated with Dendroctonus bark beetles.     

Management of palm oil mill effluent through production of cellulases by filamentous fungi

A laboratory scale study to evaluate the potentiality of filamentous fungi for the production of cellulolytic enzymes using palm oil mill effluent (POME) as a basal medium was initiated. A total of 25 filamentous fungi in which 16 filamentous fungi were isolated and purified from oil palm industrial residues and 9 strains from laboratory stock were screened using POME with 1% total suspended solids. Trichoderma reesei RUT C-30 was identified as a potential strain for cellulolytic enzyme production as compared to other genera of Aspergillus, Penicillum, Rhizopus, Phanerochaete, Trichoderma and basidiomycete groups. The results showed that T. reesei RUT C-30 gave the highest filter paper cellulase and carboxy methyl cellulase activity of 0.917 and 2.51 U/ml respectively at day 5 of fermentation. Other parameters such as growth formation, pH, filterability and total biosolids were observed to evaluate the bioconversion process.     

Optimization of conditions for the production of lignocellulolytic enzymes by Sphingobacterium sp. ksn-11 utilizing agro-wastes under submerged condition

Abstract

The present work was aimed at studying the production of lignocellulolytic enzymes, namely cellulase, xylanase, pectinase, mannanase, and laccase by a newly isolated bacterium Sphingobacterium sp. ksn-11, utilizing various agro-residues as a substrate under submerged conditions. The production of lignocellulolytic enzymes was found to be maximum at the loading of 10%(w/v) agro-residues. The enzyme secretion was enhanced by two-fold at 2?mM CaCO_3, optimum pH 7, and temperature 40°. The Field Emission Gun-Scanning Electron Microscope (FEG-SEM) results have shown the degradative effect of lignocellulases; cellulase, xylanase, mannanase, pectinase, and laccase on corn husk with 3.55?U/ml, 79.22?U/ml, 12.43?U/ml, 64.66?U/ml, and 21.12?U/ml of activity, respectively. The hydrolyzed corn husk found to be good adsorbent for polyphenols released during hydrolysis of corn husk providing suitable conditions for stability of lignocellulases. Sphingobacterium sp. ksn is proved to be a promising candidate for lignocellulolytic enzymes in view of demand for enzymes in the biofuel industry.     

Optimization of cellulase production in batch fermentation byTrichoderma reesei

Maximum cellulase production was sought by comparing the activities of the cellulases produced by differentTrichoderma reesei strains andAspergillus niger. Trichoderma reesei Rut-C30 showed higher cellulase activity than otherTrichoderma reesei strains andAspergillus niger that was isolated from soil. By optimizing the cultivation condition during shake flask culture, higher cellulase production could be achieved. The FP (filter paper) activity of 3.7 U/ml and CMCase (Carboxymethylcellulase) activity of 60 U/ml were obtained from shake flask culture. When it was grown in 2.5L fermentor, where pH and DO levels are controlled, the Enzyme activities were 133.35 U/ml (CMCase) and 11.67 U./ml (FP), respectively. Ammonium sulfate precipitation method was used to recover enzymes from fermentation broth. The dried cellulase powder showed 3074.9 U/g of CMCase activity and 166.7 U/g of FP activity with 83.5% CMCase recovery.     

麦长管蚜唾液中几种酶的鉴定、活力测定与功能分析

用Parafilm膜夹营养液法,以两种食料介质饲喂麦长管蚜Macrosiphum avenae 3龄若蚜并收集其唾液,对唾液中的酶类进行了鉴定、活力测定和功能分析。结果表明,在20%蔗糖介质提取液中,鉴定有果胶酶、多酚氧化酶和纤维素酶; 在水介质提取液中鉴定有纤维素酶; 两种介质提取液中都未鉴定出过氧化物酶。酶活力测定结果表明, 在20%蔗糖介质提取液中, 每30头蚜虫分泌的果胶酶、多酚氧化酶和纤维素酶的酶活力分别为2.59×10^-3 U/g、7×10^-3 U/g和7.89×10^-3 U/g; 在水介质提取液中,纤维素酶活力为3.68×10^-3 U/g。行为反应试验结果表明,果胶酶处理麦苗的挥发物组分能引起麦长管蚜寄生性天敌燕麦蚜茧蜂Aphidius avenae和捕食性天敌七星瓢虫Coccinella septempunctata 的嗅觉偏好反应,因此,果胶酶在麦长管蚜取食诱导小麦植株的间接防御反应中具有重要作用。     

Production of cellulases and saccharification of lignocellulosics by A. Micromonospora sp.

A locally isolated strain of Micromonospora sp. when grown on different natural cellulosic substrates gave the highest activity of carboxymethylcellulase (34 U/ml) and Avicelase (0.9 U/ml) on rice straw. Sugar cane bagasse was also a good substrate for growth and cellulase production. With commercial cellulosic substrates, highest carboxymethylcellulase (90 U/ml) and Avicelase (2.8 U/ml) activities were when the organism grew on xylan. Saccharification of sugar cane bagasse and rice straw by enzyme preparations of the organism grown on the respective substrates released 5.6 and 5.8 mg reducing sugar/ml. With all enzyme preparations, bagasse was more easily saccharified than rice straw.The authors are with the Atomic Energy Research Establishment, GPO Box 3787, Dhaka 1000, Bangladesh; N.A. Chowdhury, M. Moniruzzaman, and N. Choudhury in the Institute of Food and Radiation Biology, and N. Nahar in the Institute of Nuclear Science and Technology.     

Cellulase from Bacillus licheniformis and Brucella sp. isolated from mangrove soils of Mahanadi river delta,Odisha, India

In the present study, two cellulose-degrading bacteria (CDB-5 and CDB-12) were isolated from mangrove soils of Mahanadi river delta, based on halo zone formation in Congo red agar medium and evaluation for cellulase production in CMC broth medium. Based on morphological, biochemical and 16S rRNA gene sequencing, the two strains, CDB-5 and CDB-12, were identified as Brucella sp. and Bacillus licheniformis, respectively. The gene bank accession number of the strains CDB-5 and CDB-12 are KR632646 and KR632645, respectively. The strain Brucella sp. and B. licheniformis showed an enzyme activity of 96.37?U/ml and 98.25?U/ml, respectively, after 72?h of incubation period. Enzyme production was optimized under different growth conditions such as pH, temperature, agitation rate, carbon source, sodium chloride (NaCl), and nitrogen sources. Maximum cellulase production by both the strains was obtained in the same parameter condition such as pH (7.0), rpm (150), and NaCl (2%, w/v) which varies for other parameters. The strain, CDB-5, produced maximum cellulase at 35?°C temperature, maltose as a carbon source, and yeast extract as a nitrogen source where as the strain CDB-12 produces maximum cellulase at 45?°C temperature, carboxyl methyl cellulose (CMC) as carbon source and trypton as a nitrogen source. The bacterial crude enzyme was purified by ammonium sulfate precipitation followed by overnight dialysis. SDS-PAGE analysis of the partially purified cellulase enzyme exhibited band sizes of approximately 55 and 72?kDa.     

Addition of aluminum oxide microparticles to Trichoderma viride My preculture enhances cellulase production and influences fungal morphology

Morphological engineering techniques have recently become popular, since they are used to increase the production of a variety of metabolites and enzymes when fungi are grown in submerged cultures. This study aimed to facilitate cellulase production by adding aluminum oxide to Trichoderma viride My precultures.

The results showed that the highest cellulase activity was achieved when aluminum oxide at 10 g/L was used, and the activities of cellulase for filter paper and endoglucanase activity assays increased from 519.11 to 607.35 U/mL by 17.1%, and from 810.08 U/mL to 917.59 U/mL by 13.3%, compared with the control, respectively. Addition of aluminum oxide decreased the size of T. viride My pellets and increased the final pH. The changes in pellet diameter after the addition of different concentrations of aluminum oxide were fitted using a modified exponential decay model, which could precisely predict the pellet size by controlling aluminum oxide concentration.

The optimum concentration of microparticles, and therefore pellet size, could significantly improve cellulase production, which is an encouraging step towards commercial cellulase production.

     

桂林岩溶石山檵木群落不同恢复阶段凋落物层酶对凋落物分解的影响

该文选取桂林岩溶石山檵木群落不同恢复阶段(灌木阶段、乔灌阶段和小乔林阶段)作为研究对象,探究凋落物层酶对凋落物分解速率的影响。结果表明:不同恢复阶段凋落物经1 a分解后,凋落物剩余率分别为灌木阶段(59.58%)、乔灌阶段(61.79%)和小乔林阶段(62.02%)。不同恢复阶段凋落物分解速率随演替的进行而减小。3个不同恢复阶段凋落物层多酚氧化酶、脲酶、蔗糖酶活性均在12月份最低,多酚氧化酶活性均在3月份最高,脲酶和蔗糖酶活性均在6月份最高。3个恢复阶段纤维素酶活性变化规律趋势一致,均在6月份酶活性最高,灌木阶段纤维素酶活性在3月份最低,乔灌阶段和小乔林阶段纤维素酶活性均在9月份最低。3个不同恢复阶段的凋落物层酶活性在不同时期均表现为蔗糖酶脲酶纤维素酶多酚氧化酶。不同恢复阶段凋落物层酶活性对凋落物分解速率影响不同。灌木阶段凋落物层蔗糖酶活性与分解速率呈显著正相关(P 0.05),乔灌阶段脲酶活性与分解速率呈显著正相关(P 0.05),小乔林阶段各酶活性与分解速率相关不显著。蔗糖酶、脲酶和多酚氧化酶是影响灌木阶段凋落物分解速率的重要因素,脲酶、纤维素酶和多酚氧化酶是影响乔灌和小乔林阶段分解速率的重要因素。     

Enhancement of L-glutamate oxidase production in liquid fermentation of Streptomyces sp. by calcium addition

Summary The effect of calcium ions on production of extracellular L-glutamate oxidase (GluOx) in liquid fermentation of Streptomyces sp. N1 was investigated. By supplementing a relatively large amount of Ca²⁺ (15–40 mM) to the medium, the GluOx production was significantly enhanced, although the microbial growth was inhibited to some degree. For the first time, the highest production (i.e. 6.5 U/ml) and productivity (i.e. 0.25 U/ml/h) of GluOx as ever reported (i.e., 1.2 U/g medium after 144 h solid state fermentation and 0.59 U/ml after 96 h liquid fermentation) were achieved with addition of 30 mM Ca²⁺.     

A major symbiont shift supports a major niche shift in a clade of tree-killing bark beetles

1. One small clade of bark beetles, out of thousands of species worldwide, has shifted from using phloem to using a combination of phloem and outer bark, or to completely using outer bark. 2. The shift to outer bark has been accompanied by a shift to non-typical bark beetle mutualist fungi in Entomocorticium (Basidiomycota) and Ceratocystiopsis (Ascomycota). 3. This study compared the growth and metabolic capabilities of fungi associated with a nearly phloem-independent species, Dendroctonus brevicomis, with those of mutualist fungi of Dendroctonus ponderosae, a completely phloem-colonising beetle in a sister clade associated with more typical Ascomycota in Grosmannia and Ophiostoma. 4. Only the basidiomycete associated with D. brevicomis could degrade cellulose and lignin, whereas both the ascomycete and basidiomycete could grow in outer bark. Ascomycetes associated with D. ponderosae could not degrade cellulose or lignin or grow in outer bark. 5. Beetles and fungi in these mutualisms may best be considered as co-niche constructors. For niche construction, one organism must modify a resource in a way that enhances its fitness while also influencing fitness of other organisms using the resource. Here, the beetles kill the tree, transport the fungi into the tree, and modify the woody substrate for use by the fungi. They have also evolved mycangia to ensure vertical dissemination of the fungi. In turn, the fungi modify tree tissues and provision nutrients to the host and have evolved traits that support their acquisition and transport by the beetle host in mycangia.     

Optimization of cellulase production by <Emphasis Type="Italic">Aspergillus nidulans</Emphasis>: application in the biosoftening of cotton fibers

The enhancement of the cellulase activity of Aspergillus nidulans by combinational optimization technique and the usage of cellulase for the biofinishing of cotton fibers were investigated in this study. The strain isolated from decayed, outer shell of Arachis hypogaea was compared for the first time for its ability to produce cellulolytic enzyme in shaken cultures using the optimized media formulated by combinational statistical approach using one factor at a time methodology (OFAT), Plackett Burmann Methodology (PB) and response surface methodology (RSM). A four-factor-five-level central composite design (CCD) was employed to determine the maximum activity of cellulase at optimum levels of carboxy methyl cellulose (CMC), ammonium nitrate and potassium dihydrogen phosphate at varying pH values. The cellulase activity is the best so far obtained with this strain of Aspergillus nidulans. The optimum values of the parameters studied were found to be 0.75 mg/l, 1.5 mg/l, 0.01 mg/l, and 2.15 g/l for KH₂PO₄, NH₄NO₃, Thiamine HCl and CMC, respectively at pH 6.0. This optimization led to the fine tuning of the cellulase production, thereby enhancing the cellulase activity from 4.91 to 60.54 U/ml. This cellulase of higher activity was employed in the biofinishing of the cotton fibers. The results of the scanning electron microscope (SEM) analysis after the treatment favored the fact that maximum surface finishing was achieved at a cotton fiber concentration of 15% (w/v) at 45°C and pH 5.0 using cellulase (60.54 U/ml) at 16th hour of the treatment. A probable mechanism of enzymatic finishing of cotton fibers has also been represented.     

Temperature effects on growth of fungal symbionts of the western pine beetle,Dendroctonus brevicomis

Differences in temperature ranges and optima among poikilothermic partners in symbioses can have profound effects on their interactions and stability. In this study, we investigated how the two mutualist mycangial fungi (Ceratocystiopsis brevicomi and Entomocorticium sp. B) associated with the western pine beetle, Dendroctonus brevicomis, respond to temperature in vitro. Little variability in growth rate at the various temperatures tested occurred among isolates of C. brevicomi either within or among sites. In contrast, E. sp. B exhibited highly variable responses to mid-range temperatures among sites and within some sites, and, unlike C. brevicomi, grew not at all or only very poorly at the highest and lowest temperatures tested. This variability affected both optimal temperature and maximum growth rate. The high variability in response to some temperatures among isolates of E. sp. B in some populations indicates that the ability to capture spatial and nutritional resources can vary greatly within this species which may have considerable impact on the outcome of both inter- and intra-specific competition among the fungi within trees and the short- and long-term dynamics of the fungi with the host beetle.     

Experimental evidence of bark beetle adaptation to a fungal symbiont

The importance of symbiotic microbes to insects cannot be overstated; however, we have a poor understanding of the evolutionary processes that shape most insect–microbe interactions. Many bark beetle (Coleoptera: Curculionidae, Scolytinae) species are involved in what have been described as obligate mutualisms with symbiotic fungi. Beetles benefit through supplementing their nutrient‐poor diet with fungi and the fungi benefit through gaining transportation to resources. However, only a few beetle–fungal symbioses have been experimentally manipulated to test whether the relationship is obligate. Furthermore, none have tested for adaptation of beetles to their specific symbionts, one of the requirements for coevolution. We experimentally manipulated the western pine beetle–fungus symbiosis to determine whether the beetle is obligately dependent upon fungi and to test for fine‐scale adaptation of the beetle to one of its symbiotic fungi, Entomocorticium sp. B. We reared beetles from a single population with either a natal isolate of E. sp. B (isolated from the same population from which the beetles originated), a non‐natal isolate (a genetically divergent isolate from a geographically distant beetle population), or with no fungi. We found that fungi were crucial for the successful development of western pine beetles. We also found no significant difference in the effects of the natal and non‐natal isolate on beetle fitness parameters. However, brood adult beetles failed to incorporate the non‐natal fungus into their fungal transport structure (mycangium) indicating adaption by the beetle to particular genotypes of symbiotic fungi. Our results suggest that beetle–fungus mutualisms and symbiont fidelity may be maintained via an undescribed recognition mechanism of the beetles for particular symbionts that may promote particular associations through time.     

Evaluation of cellulases produced from four fungi cultured on furfural residues and microcrystalline cellulose

Four fungal strains—Trichoderma viride, Aspergillus niger, Trichoderma koningii, and Trichoderma reesei—were selected for cellulase production using furfural residues and microcrystalline cellulose (MCC) as the substrates. The filter paper activity (FPA) of the supernatant from each fungus was measured, and the performance of the enzymes from different fungal strains was compared. Moreover, the individual activities of the three components of the cellulase system, i.e., β-glucosidase, endoglucanase, and exoglucanase were evaluated. T. koningii showed the highest activity (27.81 FPU/ml) on furfural residues, while T. viride showed an activity of 21.61 FPU/ml on MCC. The FPA of the crude enzyme supernatant from T. koningii was 30% higher on furfural residues than on MCC. T. koningii and T. viride exhibited high stability and productivity and were chosen for cellulases production. The crystallinity index (CrI) of the furfural residues varied after digested by the fungi. The results indicated differences in the functioning of the cellulase system from each fungus. In the case of T. koningii, T. reesei and T. viride, furfural residues supported a better environment for cellulase production than MCC. Moreover, the CrI of the furfural residues decreased, indicating that this material was largely digested by the fungi. Thus, our results suggest that it may be possible to use the cellulases produced from these fungi for the simultaneous saccharification and fermentation of lignocellulosic materials in ethanol production.     

Enhanced production of cellulase usingAcidothermus cellulyticus in fed-batch culture

Summary Fed-batch fermentations of Acidothermus cellulolyticus utilizing mixtures of cellulose and sugars were investigated for potential improvements in cellulase enzyme production. In these fermentations, we combined cellulose from several sources with various simple sugars at selected concentrations. The best source of cellulose for cellulase production was found to be ball-milled Solka Floc at 15 g/l. Fed-batch fermentations with cellobiose and Solka Floc increased cell mass only slightly, but succeeded in significantly enhancing cellulase synthesis compared to batch conditions. Maximum cellulase activities obtained from fermentations initiated with 2.5 g cellobiose/l and 15 g Solka Floc/l were 0.187 units (U)/ml, achieved by continuous feeding to maintain <0.1 g cellobiose/l, and 0.215 U/ml using the same initial medium when 2.5 g cellobiose/l was step-fed after the sugar was nearly consumed. In batch, dual-substrate systems consisting of simple sugars with Solka Floc, substrate inhibition was evident in terms of specific growth rates, specific productivity values, and maximum enzyme yields. Limiting concentrations of glucose or sucrose at 5 g/l, and cellobiose at 2.5 g/l, in the presence of Solka Floc, yielded cellulase activities of 0.134, 0.159, and 0.164 U/ml, respectively. Offprint requests to: M. E. Himmel     

A new role for veratryl alcohol: regulation of synthesis of lignocellulose-degrading enzymes in the ligninolytic ascomyceteous fungus, Botryosphaeria sp.; influence of carbon source

A new physiological role for veratryl alcohol in fungi important in the biodegradation of the lignified plant cell wall is presented. Botryosphaeria sp., grown on starch, pectin, cellulose or xylan produced amylase, pectinase, cellulase, xylanase and laccase, whereas glucose and xylose repressed the synthesis of cellulase and xylanase, but not laccase. When cultured on each of these substrates in the presence of veratryl alcohol, laccase activity increased but the activities of amylase, pectinase, cellulase and xylanase significantly decreased. Basal medium containing softwood kraft lignin in the presence of veratryl alcohol induced laccases above constitutive levels. Ethyl alcohol also stimulated laccase production.     

Fungal isolates from natural pectic substrates for polygalacturonase and multienzyme production

Pectin rich wastes and waste dump yard soils were screened and eighty pectinolytic fungal isolates were obtained by enrichment culturing and ruthenium red plate assay. Eight isolates with higher zones of pectin hydrolysis were selected and tested for polygalacturonase production. One isolate identified as Aspergillus awamori MTCC 9166 with highest polygalacturonase activity was tested for utilization of raw pectins for enzyme production. Polygalacturonase production was high in raw pectin sources like Orange peel (16.8 U/ml) Jack fruit rind (38 U/ml) Carrot peel (36U/ml) and Beet root peel (24U/ml). Selected Aspergillus awamori MTCC 9166 was found to be having good polygalacturonase, xylanase, cellulase and weak amylase and protease activities. This isolate with multi-enzyme production could have application for enzymes production and degradation of fruit and vegetable waste in the process of urban waste disposal.     

Production of cellulases by thermophilic fungi grown on Leptochloa fusca straw

Seven indigenous thermophilic fungi were screened for cellulase and xylanase production when grown on Leptochloa fusca (kallar grass) straw. Aspergillus fumigatus produced the highest activities of 0.4, 2.5, 3.5 and 0.14 U/ml of filter paper cellulase, CM-cellulase, xylanase and -xylosidase, respectively. Sporotrichum thermophile produced 0.47 -glucosidase/ml. Chaetomium thermophile, Humicola grisea and Torula thermophila had lower activities than the other thermophilic fungi.The authors are with the National Institute for Biotechnology & Genetic Engineering, P.O. Box 577, Faisalabad, Pakistan.