Improving low water activity and desiccation tolerance of the biocontrol agent Pantoea agglomerans CPA-2 by osmotic treatments

AIMS: To study the improvement of tolerance to low water activity (aw) and desiccation during spray drying in Pantoea agglomerans cells subjected to mild osmotic stress during growth. METHODS AND RESULTS: The micro-organism was cultured in an unmodified liquid (control) or in aw-modified media, and viability of these cells was evaluated on unstressed (0.995) and 0.96 aw stressed solid media, in order to check total viability and aw stress tolerance respectively. Significant improvements in viability on unmodified medium were observed with cells grown for 24 h in NaCl 0.98 aw, glycerol 0.98 aw and 0.97 aw and for 48 h in NaCl 0.98 aw and 0.97 aw modified media. Both yield improvements and water stress tolerance were achieved with low aw media. Cells grown for 24 h in NaCl 0.98 aw or for 48 h in NaCl 0.98 aw, 0.97 aw and 0.96 aw, glucose 0.97 aw and glycerol 0.97 aw showed improved aw stress tolerance in comparison with control cells. The best results were obtained with NaCl treatments (0.98 aw and 0.97 aw) which also exhibited better survival rates than control cells during spray-drying process and maintained their efficacy against postharvest fungal pathogens in apples and oranges. CONCLUSIONS: NaCl treatments are very appropriate for improving P. agglomerans low aw tolerance obtaining high production levels and maintaining biocontrol efficacy. SIGNIFICANCE AND IMPACT OF THE STUDY: Improving stress tolerance of biocontrol agents could be an efficient way to obtain consistency and maintain efficacy of biological control under practical conditions.     

Accumulation of the compatible solutes, glycine-betaine and ectoine, in osmotic stress adaptation and heat shock cross-protection in the biocontrol agent Pantoea agglomerans CPA-2

AIMS: The effect of modifying the water activity (a(w)) of Pantoea agglomerans growth medium with the ionic solute NaCl on water stress resistance, heat-shock survival and intracellular accumulation of the compatible solutes glycine-betaine and ectoine were determined. METHODS AND RESULTS: The bacterium was cultured in an unmodified liquid medium or that modified with NaCl to 0.98 and 0.97 a(w), and viability of cells evaluated on a 0.96 a(w)-modified solid media to check water stress tolerance. Cells grown under ionic stress had better water stress tolerance than control cells. These cells also had cross-protection to heat stress (30 min, 45 degrees C). The modified cells accumulated substantial amounts of the compatible solutes glycine-betaine and ectoine in contrast to the control cells, which contained little or none of these two compounds. CONCLUSIONS: Improvement in osmotic and thermal tolerance of cells of the biocontrol agent P. agglomerans by modifying growth media with the ionic solute NaCl was achieved. The compatible solutes glycine-betaine and ectoine play a critical role in environmental stress tolerance improvement. SIGNIFICANCE AND IMPACT OF THE STUDY: This approach provides a method for improving the physiological quality of inocula and could have implications for formulation and shelf-life of biocontrol agents.     

拮抗木霉耐多菌灵菌株的筛选

该文进行了耐多菌灵生防木霉菌株的筛选和生防效果的测定研究。为获得对灰葡萄孢霉等病原菌有显著拮抗能力的耐多菌灵木霉菌株,通过含药平板诱导和紫外线诱变,获得5株耐药性菌株,在多菌灵2.0g/L浓度下生长较好,Ec50值提高了40多倍;抗性菌株在含药培养基上连续继代转移培养10次,抗性程度未见下降;经过平板对峙和活体拮抗性测定,确定T42-2为最佳拮抗性耐多菌灵菌株,其对黄瓜灰霉病的防效达92.13%,并且对多菌灵、速克灵和三唑铜存在交互抗性。     

Osmotic stress adaptation, compatible solutes accumulation and biocontrol efficacy of two potential biocontrol agents on Fusarium head blight in wheat

Fusarium head blight (FHB) caused by Gibberella zeae (anamorph = Fusarium graminearum) is a devastating disease that causes extensive yield and quality losses to wheat in humid and semi-humid regions of the world. Biological control has been demonstrated to be effective under laboratory conditions but a few biocontrol products have been effective under field conditions. The improvement in the physiological quality of biocontrol agents may improve survival under field conditions, and therefore, enhance biocontrol activity. Bacillus subtilis RC 218 and Brevibacillus sp. RC 263 were isolated from wheat anthers and showed significant effect on control of FHB under greenhouse assays. This study showed the effect of water availability measured as water activity (a_W) using a growth medium modified with NaCl, glycerol and glucose on: (i) osmotic stress tolerance, (ii) viability in modified liquid medium, (iii) quantitative intracellular accumulation of betaine and ectoine and (iv) the biocontrol efficacy of the physiologically improved agents. Viability of B. subtilis RC 218 in NaCl modified media was similar to the control. Brevibacillus sp. RC 263 showed a limited adaptation to growth in osmotic stress. Betaine was detected in high levels in modified cells but ectoine accumulation was similar to the control cells. Biocontrol activity was studied in greenhouse assays on wheat inoculated at anthesis period with F. graminearum RC 276. Treatments with modified bacteria reduced disease severity from 60% for the control to below 20%. The physiological improvement of biocontrol agents could be an effective strategy to enhance stress tolerance and biocontrol activity under fluctuating environmental conditions.     

Colonization of wheat roots from seed‐applied spores of Idriella (Microdochium) bolleyi: A biocontrol agent of take‐all

Wheat seedlings were grown in containers of perlite in a glasshouse, and spores of Idriella bolleyi were applied either to the seeds in alginate gel or to the perlite as aqueous suspensions. Growth and sporulation of the fungus on seeds and roots were assessed by plating methods and by retrieval of spores from water that drained from the plant containers. Idriella bolleyi sporulated heavily on seeds for up to 3 weeks when applied to them in alginate gel, andmost of the root system was infected from newly formed spores that were carried in percolating water. Removal of the inoculated seeds from seedlings at 7 days reduced the degree of root infection and temporarily reduced the number of spores in drainage water; however, spore numbers increased to control levels (seeds attached) by 21 days, indicating subsequent sporulation on the roots. When spores were applied to perlite containing young (0–3 days) seedlings I. bolleyi colonized the seeds, sporulated heavily on them and extensively colonized the roots. However, it established poorly on seeds and roots if added to the perlite when seedlings were 11 days or older. The results suggest that early growth on seeds is necessary for establishment of a high population of I. bolleyi, and that cycles of sporulation on seeds and then roots contribute to colonization of the rhizosphere. The features associated with the rhizosphere competence of I. bolleyi are compared with those for systemic colonization of the xylem by vascular wilt fungi, and suggest a new approach to the selection of root‐colonizing biocontrol agents.     

Predicted ecological niches and environmental resilience of different formulations of the biocontrol yeast <Emphasis Type="Italic">Candida sake</Emphasis> CPA-1 using the Bioscreen C

Environmental resilience of biocontrol microorganisms has been a major bottleneck in the development of effective formulations. Candida sake is an effective biocontrol agent (BCA) against Penicillium expansum, Botrytis cinerea or Rhizopus stolonifer, and different formulations of the BCA have been optimised recently. The objective of this study was to compare the relative tolerance of different dry and liquid formulations of the biocontrol yeast C. sake CPA-1 to interacting environmental conditions using the Bioscreen C. Initially, the use of this automated turbidimetric method was optimised for use with different formulations of the biocontrol yeast. The best growth curves were obtained for the C. sake CPA-1 strain when grown in a synthetic grape juice medium under continuous shaking and with an initial concentration of 10⁵ CFUs ml^?1. All the formulations showed a direct relationship between optical density values and yeast concentrations. Temperature (15–30 °C) and water activity (a_w; 0.94–0.99) influenced the yeast resilience most profoundly, whereas the effect of pH (3–7) was minimal. In general, the liquid formulation grew faster in more interacting environmental conditions but only the yeast cells in the dry potato starch formulation could grow in some stress conditions. This rapid screening method can be used for effective identification of the resilience of different biocontrol formulations under interacting ecological abiotic conditions.     

Osmotic stress adaptation,compatible solutes accumulation and biocontrol efficacy of two potential biocontrol agents on Fusarium head blight in wheat

Fusarium head blight (FHB) caused by Gibberella zeae (anamorph = Fusarium graminearum) is a devastating disease that causes extensive yield and quality losses to wheat in humid and semi-humid regions of the world. Biological control has been demonstrated to be effective under laboratory conditions but a few biocontrol products have been effective under field conditions. The improvement in the physiological quality of biocontrol agents may improve survival under field conditions, and therefore, enhance biocontrol activity. Bacillus subtilis RC 218 and Brevibacillus sp. RC 263 were isolated from wheat anthers and showed significant effect on control of FHB under greenhouse assays. This study showed the effect of water availability measured as water activity (a_W) using a growth medium modified with NaCl, glycerol and glucose on: (i) osmotic stress tolerance, (ii) viability in modified liquid medium, (iii) quantitative intracellular accumulation of betaine and ectoine and (iv) the biocontrol efficacy of the physiologically improved agents. Viability of B. subtilis RC 218 in NaCl modified media was similar to the control. Brevibacillus sp. RC 263 showed a limited adaptation to growth in osmotic stress. Betaine was detected in high levels in modified cells but ectoine accumulation was similar to the control cells. Biocontrol activity was studied in greenhouse assays on wheat inoculated at anthesis period with F. graminearum RC 276. Treatments with modified bacteria reduced disease severity from 60% for the control to below 20%. The physiological improvement of biocontrol agents could be an effective strategy to enhance stress tolerance and biocontrol activity under fluctuating environmental conditions.     

生防真菌分生孢子抗氧化能力与多糖含量的关系

作为重要的丝孢类昆虫病原真菌,球孢白僵菌和玫烟色棒束孢因其易于生产和环境友好等优点而在害虫生防防治中受到广泛青睐。为初步探求孢子耐氧化力及其与孢子多糖含量的关系,球孢白僵菌和玫烟色棒束孢11株菌经胁迫后的残存指数随氧化剂H2O2浓度增加而减小。所有菌株的残存指数均能良好地与Logistic方程拟合,并计算出各菌株在氧化胁迫条件下的半致死浓度。结果显示玫烟色棒束孢孢子的耐氧化力强于球孢白僵菌。两种真菌的分生孢子耐氧力与各自多糖含量呈现良好线性正相关。培养基碳源成分和浓度变化可影响球孢白僵菌孢子耐氧化力,但耐氧化力与多糖含量依旧呈现线性正相关。由此可见,生防真菌分生孢子的耐氧化力的确与多糖积累有关,并在一定程度上受培养条件的调节。研究结果有望为提高生防真菌孢子环境稳定性提供新的策略。     

The role of lager beer yeast in oxidative stability of model beer

Aims: In this study, we investigated the relationship between the ability of lager brewing yeast strains to tolerate oxidative stress and their ability to produce oxidative stable model beer. Methods and Results: Screening of 21 lager brewing yeast strains against diamide and paraquat showed that the oxidative stress resistance was strain dependent. Fermentation of model wort in European Brewing Convention tubes using three yeast strains with varying oxidative stress resistances resulted in three model beers with different rates of radical formation as measured by electron spin resonance in forced ageing experiments. Interestingly, the strain with the lowest oxidative stress resistance and lowest secretion of thioredoxin, as measured by Western blotting, resulted in the highest uptake of iron, as measured by inductively coupled plasma‐mass spectrometry, and the slowest formation of radicals in the model beers. Conclusions: A more oxidative stable beer is not obtained by a more‐oxidative‐stress‐tolerant lager brewing yeast strain, exhibiting a higher secretion of thioredoxin, but rather by a less‐oxidative‐stress‐tolerant strain, exhibiting a higher iron uptake. Significance and Impact of the Study: To obtain lager beers with enhanced oxidative stability, yeast strains should be screened for their low oxidative stress tolerance and/or high ability to take up iron rather than for their high oxidative stress tolerance and/or high ability to secrete thioredoxin.     

松萝内生酿酒酵母菌株耐酸生理特性与分子机制探究

【目的】对我国西藏地区来源的不同酵母菌株进行有机酸发酵性能测试,此外,对具有良好产酸性能的分离自松萝内部的酿酒酵母菌株Saccharomy cescerevisiae 2-2进行耐酸性能分析,并探究其耐酸较强的分子机制。【方法】比较不同糖浓度培养基液体发酵培养过程中pH的变化,并比较低pH胁迫条件下菌株的生长,检测酿酒酵母菌株的产酸潜力和耐酸特性;对菌株2-2和模式酵母菌株S288C进行比较基因组分析,并利用实时荧光定量聚合酶链式反应(real-time fluorescence quantitative polymerase chain reaction,RT-qPCR)分析关键基因的转录,探究菌株2-2耐酸分子机制。【结果】松萝内生酿酒酵母2-2在所有检测的菌株中产酸潜力较大,耐酸性能较好。在菌株2-2中与胁迫耐受性相关的基因PDR15、PDR12和SUR1在低pH胁迫条件下存在显著的上调或下调,但这些基因转录变化趋势与菌株S288C相反。【结论】松萝内生酿酒酵母2-2是一株产酸耐酸性能较好的菌株,对其独特的调节机制进行深入分析,有希望选育性能更好的产酸酵母菌株。     

Screening of freeze-dried protective agents for the formulation of biocontrol strains, Bacillus cereus AR156, Burkholderia vietnamiensis B418 and Pantoea agglomerans 2Re40

Aims: The effects of different freeze‐drying protective agents on the viabilities of biocontrol strains Bacillus cereus AR156, Burkholderia vietnamiensis B418 and Pantoea agglomerans 2Re40 were investigated. Method and Results: Several concentrations of protective and rehydration media were tested to improve the survival of biocontrol agents after freeze‐drying. The subsequent survival rates during storage and rehydration media of freeze‐dried biocontrol strains were also examined. Conclusions: The results indicated that cellobiose (5%) and d ‐galactose (5%) gave maximum viability of strains Bu. vietnamiensis B418 and P. agglomerans 2Re40 (98 and 54·3% respectively) while the perfect one (100%) of strain B. cereus AR156 was obtained with sucrose (5%) during freeze‐drying, and the highest survival of the three strains was reached when they were rehydrated with 10% nonfat skim milk. In the following storage, the survival rates showed that B. cereus AR156 could still reach 50% after 12 months. Significance and Impact of the study: This study showed that freeze‐drying could be used to stabilize cells of these three biocontrol strains. Further studies should focus on the scale‐up possibilities and formulation development.     

Diversity and salt tolerance of native Acacia rhizobia isolated from saline and non‐saline soils

Re‐establishing native vegetation in stressed soils is of considerable importance in many parts of the world, leading to significant interest in using plant–soil symbiont interactions to increase the cost‐effectiveness of large‐scale restoration. However, effective use of soil microbes in revegetation requires knowledge of how microbe communities vary along environmental stress gradients, as well as how such variation relates to symbiont effectiveness. In Australia, shrubby legumes dominate many ecosystems where dryland salinity is a major issue, and improving plant establishment in saline soils is a priority of regional management agencies. In this study, strains of rhizobial bacteria were isolated from a range of Acacia spp. growing in saline and non‐saline soils. Replicates of each strain were grown under several salinity levels in liquid culture and characterized for growth and salt tolerance. Genetic characterization of rhizobia showed considerable variation among strains, with salt tolerance and growth generally higher in rhizobial populations derived from more saline soils. These strains showed markedly different genetic profiles and generic affiliations to those from more temperate soils, suggesting community differentiation in relation to salt stress. The identification of novel genomic species from saline soils suggests that the diversity of rhizobia associated with Australian Acacia spp. is significantly greater than previously described. Overall, the ability of some symbiotically effective strains to tolerate high salinity is promising with regard to improving host plant re‐establishment in these soils.     

Selection of newly isolated mushroom strains for tolerance and biosorption of zinc in vitro

Nine newly isolated mushroom strains were tested to assess both their zinc tolerance and potential for zinc removal from an aqueous solution. Four strains of ectomycorrhizal fungi, namely Clavariadelphus truncatus (T 192), Rhizopogon roseolus (T 21), Lepista nuda (T 373), and Tricholoma equestre (T 174), along with five strains of white rot fungi, Lenzites betulina (S 2), Trametes hirsuta (T 587), Ganoderma spp. (T 99), Polyporus arcularius (T 438), and Ganoderma carnosum (M 88), were investigated using zinc-amended solid and liquid media. Their biosorption properties were also determined. The colony diameter and dry weight were used as tolerance indices for fungal growth. C. truncatus and T. equestre were not strongly inhibited at the highest concentrations of (225 mg/l) zinc in solid media. The most tolerant four strains with solid media, C. truncatus, G. carnosum, T. hirsuta, and T. equestre, were then chosen for tolerance tests in liquid media. An ectomycorrhizal strain, C. truncatus, was also detected as the most tolerant strain in liquid media. However, the metal-tolerant strains demonstrated weak activity in the biosorption studies. In contrast, the highest biosorption activity was presented by a more sensitive strain, G. carnosum. In addition, seven different biosorbent types from G. carnosum (M 88) were compared for their Zn (II) biosorption in batch experiments.     

Influence of agar on in vitro cultures: I. Physicochemical properties of agar and agar gelled media

Summary The success of in vitro culture is related to several factors. Beside factors associated with the plant material or the medium composition, the physicochemical characteristics of gelled media can play an important role. In this paper, the latter aspect has been considered and the nature of agar powders has been investigated. Moreover, the process of gel formation for three different media and the availability of water and minerals for the corresponding gels have been studied. Analysis of agar powders showed that they can contain different amounts of impurities and the dialysis of these powders suggested that the impurities might be available to the tissues. Thermal analysis on the hygroscopic properties of the agar brands suggest the importance of these data to obtain comparable and reproducible gelled media. The study on the process of formation of gelled media indicates that there is a critical temperature T_ss which can be used to control the gel processing. In fact, at this temperature, agar powders in water transform into a sol status through a rapid shift of electrical conductivity. Water potential of the medium, water loss from gels over the culture period, and the ease of releasing liquid from gels under pressure were shown to be different for different agar brands. A different availability of water and minerals in Murashige and Skoog medium was deduced from the gels prepared with three agar brands (Oxoid, Merck, and Roth).     

Biodegradation of nicotine by newly isolated Pseudomonas sp. CS3 and its metabolites

Aims: Isolation and characterization of nicotine‐degrading bacteria with advantages suitable for the treatment of nicotine‐contaminated water and soil and detection of their metabolites. Methods and Results: A novel nicotine‐degrading bacterial strain was isolated from tobacco field soil. Based on morphological and physiochemical properties and sequence of 16S rDNA, the isolate was identified as Pseudomonas sp., designated as CS3. The optimal culture conditions of strain CS3 for nicotine degradation were 30°C and pH 7·0. However, the strain showed broad pH adaptability with high nicotine‐degrading activity between pH 6·0 and 10·0. Strain CS3 could decompose nicotine nearly completely within 24 h in liquid culture (1000 mg L^?1 nicotine) or within 72 h in soil (1000–2500 mg kg^?1 nicotine) and could endure up to 4000 mg L^?1 nicotine in liquid media and 5000 mg kg^?1 nicotine in soil. Degradation tests in flask revealed that the strain had excellent stability and high degradation activity during the repetitive degradation processes. Additionally, three intermediates, 3‐(3,4‐dihydro‐2H‐pyrrol‐5‐yl) pyridine, 1‐methyl‐5‐(3‐pyridyl) pyrrolidine‐2‐ol and cotinine, were identified by GC/MS and NMR analyses. Conclusions: The isolate CS3 showed outstanding nicotine‐degrading characteristics such as high degradation efficiency, strong substrate endurance, broad pH adaptability, and stability and persistence in repetitive degradation processes and may serve as an excellent candidate for applications in the bioaugmentation process to treat nicotine‐contaminated water and soil. Also, detection of nicotine metabolites suggests that strain CS3 might decompose nicotine via a unique nicotine‐degradation pathway. Significance and Impact of the Study: The advantage of applying the isolated strain lies in broad pH adaptability and stability and persistence in repetitive use, the properties previously less focused in other nicotine‐degrading micro‐organisms. The strain might decompose nicotine via a nicotine‐degradation pathway different from those of other nicotine‐utilizing Pseudomonas bacteria reported earlier, another highlight in this study.     

Nutrient depletion modifies cell wall adsorption activity of wine yeast

Yeast cell wall is a structure that helps yeasts to manage and respond to many environmental stresses. The mannosylphosphorylation is a modification in response to stress that provides the cell wall with negative charges able to bind compounds present in the environment. Phenotypes related to the cell wall modification such as the filamentous growth in Saccharomyces cerevisiae are affected by nutrient depletion. The present work aimed at describing the effect of carbon and/or nitrogen limitation on the aptitude of S. cerevisiae strains to bind coloured polyphenols. Carbon- and nitrogen-rich or deficient media supplemented with grape polyphenols were used to simulate different grape juice conditions—early, mid, ‘adjusted’ for nitrogen, and late fermentations. In early fermentation condition, the R+G+B values range from 106 (high adsorption, strain Sc1128) to 192 (low adsorption, strain Σ1278b), in mid-fermentation the values range from 111 (high adsorption, strain Sc1321) to 258 (low adsorption, strain Sc2306), in ‘adjusted’ for nitrogen conditions the values range from 105 (high adsorption, strain Sc1321) to 194 (low adsorption, strain Sc2306) while in late fermentation conditions the values range from 101 (high adsorption, strain Sc384) to 293 (low adsorption, strain Sc2306). The effect of nutrient availability is not univocal for all the strains and the different media tested modified the strains behaviour. In all the media the strains show significant differences. Results demonstrate that wine yeasts decrease/increase their parietal adsorption activity according to the nutrient availability. The wide range of strain variability observed could be useful in selecting wine starters.     

Hydraulic contribution in cell elongation of tissue-cultured plants: growth retardation induced by osmotic and temperature stresses and addition of 2,4-dichlorophenoxyacetic acid and benzylaminopurine

This work was undertaken to determine the growth parameters of Lockhart’s equation for finding which component was predominantly contributing to the cell expansion rates of plants subjected to environmental stresses under tissue-culture conditions. Embryos isolated from soybean (Glycine max [L.] Merr.) and kidney bean (Phaseolus vulgaris L.) seeds were grown under tissue-culture conditions. The water potential of culture media ranged from ? 0·02 to ? 0·94 MPa so that nutrient deficiency and salt stress conditions could be applied. Additionally, the temperature of culture conditions was set from 10 to 40 °C to apply low-temperature and high-temperature stresses on plants grown at the optimum concentration of culture medium. Cell expansion could be inhibited completely by adding 2,4-dichlorophenoxyacetic acid and benzylaminopurine to culture media to form callus tissue. The sizes of the water potential gradient between the water source and elongating cells correlated with the speed of growth rates under nutrient deficiency, salt stress, growth retardation induced by plant hormones, low-temperature and high-temperature conditions in the present study, indicating that cell expansion rates were mainly associated with how much water could be absorbed by elongating cells regardless of the kinds of environmental stress conditions applied.     

Effects of salt stress and rhizobial inoculation on growth and nitrogen fixation of three peanut cultivars

Increasing soil salinity represents a major constraint for agriculture in arid and semi‐arid lands, where mineral nitrogen (N) deficiency is also a frequent characteristic of soils. Biological N fixation by legumes may constitute a sustainable alternative to chemical fertilisation in salinity‐affected areas, provided that adapted cultivars and inoculants are available. Here, the performance of three peanut cultivars nodulated with two different rhizobial strains that differ in their salt tolerance was evaluated under moderately saline water irrigation and compared with that of N‐fertilised plants. Shoot weight was used as an indicator of yield. Under non‐saline conditions, higher yields were obtained using N fertilisation rather than inoculation for all the varieties tested. However, under salt stress, the yield of inoculated plants became comparable to that of N‐fertilised plants, with minor differences depending on the peanut cultivar and rhizobial strain. Our results indicate that N fixation might represent an economical, competitive and environmentally friendly choice with respect to mineral N fertilisation for peanut cultivation under moderate saline conditions.     

Polysaccharide-hydrolyzing enzymes ofFrankia (Actinomycetales)

Studies were made of the polysaccharide-hydrolyzing activity inFrankia (Actinomycetales) grown in synthetic media using modifications of three standard assay procedures. In screening five different strains ofFrankia for cellulase activity, based on the method of utilization of cellulose in liquid culture, only one strain, CcI3, degraded filter paper cellulose to complete disintegration and only under very specific conditions of pH and primary carbon source. When carboxymethylcellulose (CMC) at 1% was used as substrate, all five strains showed the capacity to produce reducing sugars as hydrolytic products. Microcystalline cellulose, xylans and gum arabic were hydrolyzed to a lesser extent. Optimum activity depended upon pH and primary carbon source with pH 5.0 and pyruvate or propionate producing highest activities. In fractionation studies of culturedFrankia, assays for hydrolysis of 1% CMC in liquid medium showed that highest activity was in the enzyme preparation supernatant with lesser activity in the cell-free extract and cell wall fractions.Frankia strain CpI1 showed the greatest total hydrolytic activity against CMC after 2 weeks of culture. Strains ArI3 and CcI3 also showed good activity. The agar plate method for direct dye-polysaccharide interaction proved to be the least sensitive assay method with only ArI3 showing significant activity using CMC as substrate. It appears that theFranka strains grown in synthetic media all showed hydrolytic activity but the degree of hydrolysis of polysaccharides to reducing sugars depends upon strain of bacteria and very specific cultural conditions.