魔芋白绢病病原齐整小核菌(Sclerotium rolfsii)的生物学特性与室内毒力

【目的】探明魔芋白绢病的病原微生物类型及其生物学特性,筛选高效杀菌剂,为魔芋白绢病田间综合防治措施的制订和实施提供理论依据。【方法】采用组织分离和土壤稀释法,对具有典型白绢病症状的魔芋植株和附近土壤进行病原分离和纯化,通过形态学和分子系统学鉴定病原菌;采用菌丝生长速率法探究病原的生物学特性和7种药剂(苯醚甲环唑、吡唑醚菌酯、氟硅唑、戊唑醇、噻呋酰胺、烯唑醇及丙环唑)对其室内毒力。【结果】魔芋白绢病病原为齐整小核菌(Sclerotium rolfsii),其菌丝在OA和PDA培养基生长速度最快,培养3 d,菌落直径达7 cm,菌丝生长速率为2.33 cm/d;25~30℃为最适培养温度,培养3 d,菌落直径达7.2 cm,菌丝生长速率为2.48 cm/d;pH为5~7的偏弱酸环境有利于菌丝生长,生长速率最高达2.33 cm/d;氮源以蛋白胨、碳源以可溶性淀粉利于病原菌丝生长,生长速率分别为2.14 cm/d和2.43 cm/d,显著高于其他处理。戊唑醇、烯唑醇、噻呋酰胺、苯醚甲环唑、吡唑醚菌酯和丙环唑对白绢病均具有很好的抑制作用,其中,戊唑醇的防效最好,EC_(50)为0.101 mg/L;其后依次为烯唑醇、噻呋酰胺、苯醚甲环唑、吡唑醚菌酯和丙环唑,EC_(50)依次为0.165 mg/L、0.185 mg/L、1.026 mg/L、1.842 mg/L和2.112 mg/L。【结论】S.rolfsii为引起土传病害魔芋白绢病的病原,其适宜生长温度为25~30℃,弱酸和营养丰富的环境有利于病原菌丝生长,戊唑醇可有效抑制菌丝生长。

The quantity of OA and activity of cellulase and polygalacturonase are involved in variation of virulence in Sclerotium rolfsii

In order to understand the pathogenic mechanisms of Sclerotium rolfsii on peanut and to analyze the variation of virulence in S.rolfsii strains,the highly virulent strain(ZY2)and weakly virulent strain(GP3-1)were investigated under both in vivo and in vitro conditions.The results indicated that S.rolfsii directly infected peanut by producing infection cushions.ZY2 formed infection cushions earlier than GP3-1,and ZY2 produced a greater number of infection cushions compare to GP3-1.Both strains could utilize cellulose,xylose,or polygalacturonic acid in the Czapek medium.The activities of cellulase(CL)and polygalacturonase(PG)in the inoculated peanut stems increased significantly at 9 h after inoculation.The activities of CL and PG produced by ZY2 in the inoculated stems were significantly higher than that produced by GP3-1.Both strains could produce oxalic acid(OA),and the content of OA produced by ZY2 in the inoculated stems was higher than that produced by GP3-1.In summary,it suggested that S.rolfsii destroyed peanut cells through physical and biochemical factors by secreting a large amount of OA,CL and PG during the formation of infection cushions.The difference in OA content,activity of CL and PG produced by highly and weakly virulent strains played important roles in variation of virulence.

Induced Sporophore and Sclerotium of Physarum pusillum(Myxomycetes)

[Objective]The paper was to culture the plasmodia of Physarum pusillum for obtaining the optimal condition of sporophores and sclerotium,to reduce the agricultural diseases.[Method]Liquid culture combined with oat-agar culture was used to culture plasmodia of P.pusillum.Then plasmodium was induced to obtain Sporophores in lab by hunger and adjusting light（3 000,6 000,9 000,12 000 lx）and temperature（20,22,24,26 ℃）.[Result]Sporophores and sclerotium of P.pusillum were obtained from both oat-agar media and liquid media.The optimal conditions were 26 ℃,6 000 lx and 22 ℃,3 000 lx.[Conclusion]The result provided theoretical basis for reducing the loss of crops caused by myxomycete as much as possible in agricultural production.

加拿大一枝黄花白绢病(Sclerotium rolfsii)菌株SC64的生物学特性研究

在实验室条件下初步研究了加拿大一枝黄花白绢病菌株SC64的形态特征、致病性和生物学特性。结果表明:引起加拿大一枝黄花白绢病的病原物是罗氏白绢小菌核菌(Sclerotium rolfsii Sacc.);病菌最适生长培养基为Richard固体培养基和PDA,菌丝生长温度为15～40℃,最适30℃;在偏酸性条件下生长较好,生长适宜pH值为4.0～6.0,最适为5.0;在所测试的碳源中,对蔗糖的利用最好,对乳糖和半乳糖利用最差;氮源测试结果显示对蛋白胨和硝酸钾利用最好,对尿素的利用最差。菌核萌发温度为15～40℃,最适为30℃;菌核在含水量≥50%的麸皮基质上萌发较好;适宜萌发的pH值范围为3.0～9.0。菌丝的致死温度为45℃10 min,菌核的抑制萌发温度为50℃10 min。

PREPARATION AND STRUCTURE OF FIVE DERIVATIVES OF β-(1→3)-D-GLUCAN ISOLATED FROM PORIA COCOS SCLEROTIUM

A new solvent of cellulose (1.5 mol/L NaOH/0.5 mol/L urea aqueous solution) was used as one of the homogeneous reaction media of polysaccharides for methylation, hydroxyethylation and hydroxypropylation. A water insoluble β -(1—>3)-D-glucan, sample PCS3- isolated from fresh sclerotium of Poria cocos was sulfated in dimethyl sulfoxide (Me 2 SO), carboxymethylated in NaOH, isopropanol solution, as well as methylated, hydroxyethylated and hydroxypropylated in the new solvent system, respectively, to obtain five water-soluble derivatives coded as S-PCS3- C- PCS3- M-PCS3- HE-PCS3- and HP-PCS3- Their chemical structure and distribution of substitution were characterized by infrared spectroscopy (IR), elementary analysis (EA), 1 H-NMR, 13 C-NMR, 2D-COSY, 2D-TOCSY and 2D- 1 H-detected 1H 13C HMQC spectra. The results reveal that the relative reactivity of hydroxyl groups of the β -(1-?3)-D-glucan is in the order C-6 > C-4 > C-2 on the whole. The substitution of the samples S-PCS3- C-PCS3- and M-PCS3- occurred mainly at C-6 position and secondly at C-4 and C-2 positions, and that of HE-PCS3- occurred at C-6 and C-4 positions and of HP-PCS3- almost completely occurred at C-6 position. The degrees of substitution (DS) obtained from 13 C-NMR range from 0.23 to 1.27. The water solubility of the derivatives is in the order S-PCS3- >C-PCS3- >M-PCS3- >HE-PCS3- >HP-PCS3- This work provides a novel and nonpolluting process for the methylation, hydroxyethylation and hydroxypropylation of β -(1—>3)-D-glucan.

Streptomyces sp. RP1A-12 mediated control of peanut stem rot caused by Sclerotium rolfsii

Sclerotium rolfsii Sacc. is a destructive soilborne fungal pathogen with a wide host range that includes peanuts. Biological control offers an interesting alternative to fungicides for sustainable management of soilborne diseases. The current investigation is aimed at evaluating one potential biocontrol agent Streptomyces sp. RP1 A-12 for growth promotion and the management of peanut stem rot disease caused by S. rolfsii under field conditions. Preliminary studies conducted under in vitro and the greenhouse conditions showed promising results against the stem rot pathogen. Further in vitro and pot experiments conducted to assess Streptomyces sp. RP1 A-12 for its growth promoting abilities using whole organisms have shown an increase in seed germination, root and shoot length. Other parameters like nodule number and plant biomass were also significantly increased over control treatments indicating that the test bioagent possesses growth promoting abilities along with disease suppression capabilities. Subsequently field studies were carried out for two consecutive rainy seasons. The bioagent was applied as whole organism and partially purified crude metabolites. Results indicate the bioagent reduced stem rot disease incidence by 64–67% and 22–49% respectively in two field trials conducted with notable increase in yield. Partially purified Streptomyces sp. RP1 A-12 metabolites exhibited an even greater effect in reducing the incidence and severity of stem rot compared to the pathogen inoculated control.

室内模拟旱直播稻田环境下齐整小核菌Sclerotium rolfsii菌株SC64致病力的影响因子及除草效果的研究

本文通过温室盆栽试验,以鳢肠Eclipta prostrata为例,研究杂草叶龄、不同含菌丝体基质接种量以及温度、土壤湿度、保湿期时间等环境因子对菌株SC64侵染鳢肠的影响。在此基础上,温室条件下模拟旱直播水稻田环境下菌株SC64防治节节菜Rotala indica、异型莎草Cyperus difformis、鸭舌草Monochoria vaginalis和耳叶水苋Ammannia arenaria的试验。结果表明,鳢肠幼苗在5叶期以下,接种含菌丝体固体基质剂量在100g m 2以上,温度范围27～33℃,土壤相对饱水90%左右,并且接菌后湿度保持至少24 h,是菌株SC64达到理想除草效果的必需条件。温室模拟试验结果显示,接种含菌丝体固体基质剂量在120 g m 2时,28 d可引起节节菜和鸭舌草81%和74%的植株死亡率及83.8%和82.5%的鲜重抑制率;对异型莎草和耳叶水苋的防效稍差,仅能引起约50%的植株死亡率及60%～65%的鲜重抑制率。这表明菌株SC64可有效控制阔叶杂草,具有开发作为生物除草剂的潜力。

INFLUENCE OF MOLECULAR WEIGHT AND PERIODATE-MODIFICATION OF β-D-GLUCANS FROM PORIA COCOS SCLEROTIUM ON ANTITUMOR ACTIVITIES

In this work, influence of molecular weight and periodate modification ofβ-D-glucans isolated from Poria cocos sclerotium on the antitumor activities against Sar-coma 180 and Ehrlich ascites carcinoma (EAC) tumor was studied. The results show thattwo glucans PC3 (linear β-(1→3)-D-glucan) and PC4 [β-(1→3)-D-glucan with a fewof branches and glucuronic acid] are devoid of antitumor activity. However, when theglucans were modified by periodate oxidation, borohydride reduction and mild hydrolysisor partially hydrolysis, the derivatives have obvious antitumor activities. The decreasein molecular weight of glucans after periodate modification hardly affects their antitumoractions, but on the other hand, the decrease of molecular weight without periodate modi-fication could lead to an enhancement of the antitumor activities. Moreover, the glucansand these derivatives have much higher enhancement ratios of body weight of mice thanthat of 5-Fluorouracil (5-Fu), suggesting that they are less toxic than 5-Fu.

Evaluation of Selected Chemical,Biological Fungicides,and Induced Resistance to Control White Rot(Sclerotium rolfsii Sacc.)on Tomato

Tomato(Solanum lycopersicum)is one of the most widely grown and consumed as fresh vegetable in the world.Sclerotium rolfsii Sacc.is one of the most destructive diseases and affects more than 500 plant species.This study was conducted for“Evaluation of Selected Chemical,Biological Fungicides,and Induced Resistant to Control White Rot(Sclerotium rolfsii Sacc.)on Tomato”.The experiment was conducted at the Royal University of Agriculture and divided into two sections.The first section conducted under in vitro condition consists of four treatments T1 control treatment,T2 copper hydroxide,T3 azoxystrobin+difenoconazole,T4 metalaxyl+mancozeb in Nagoya Laboratory,and had been starting from 11st May to 15th May 2020.Section two was conducted in a pot in net-house which started from 1st January 2019 to 19th February 2019 and 1st January to 19th February 2020 and arranged in RCBD(Randomized Completely Block Design)with six replications and nine treatments.T0 negative control,T1 inoculate but not treatment,T2 metalaxyl+mancozeb,T3 azoxystrobin+difenoconazole,T4 copper hydroxide,T5 acibenzolar-S-methyl,T6 chicken dung+Trichoderma harzianum,T7 acibenzolar-S-methyl+chicken dung+Trichoderma harzianum,T8 acibenzolar-S-methyl+metalaxyl+mancozeb.Data were collected on colonizing diameter in vitro.For pot experiment collected on disease incidence,incubation period Based on the result under in vitro condition metalaxyl+mancozeb highly inhibited germination of Sclerotium rolfsii when compared with another treatment.However,the application of copper hydroxide seems less effective compared with control.For in pot experiment,when treated after symptom of southern blight appeared to seem less effective on the severity of southern blight.In contrast,when applied as a protectant and curative when symptoms appeared only systemic fungicide azoxystrobin+difenoconazole significantly reduced the severity of Sclerotium rolfsii and delayed incubation period while compared with other treatments(p<0.05)and was followed by metalaxyl+mancozeb,whereas,copper hydroxide,acibenzolar-S-methyl,chicken dung+Trichoderma harzianum,acibenzolar-S-methyl+chicken dung+Trichoderma harzianum,acibenzolar-S-methyl+metalaxyl+mancozeb were less effective(p>0.05).Based on the three experiments we can assume that fungicide is more effective in reducing the growth of the pathogen and delaying the incubation period of fungal colonization when compared with biological control and induced resistance.

麝香草酚控制核盘菌侵染不结球白菜效应研究

核盘菌可侵染多种蔬菜,造成蔬菜采后品质下降,筛选高效安全的天然保鲜剂是控制核盘菌侵染的有效手段。通过离体和活体接种试验,研究了植物源天然化合物麝香草酚对核盘菌侵染青菜的控制效应。结果表明:20、40、60μg/mL麝香草酚处理后的菌丝鲜重分别比对照组显著下降了33.8%、53.7%、88.1%;菌丝干重分别比对照组下降了24.5%、53.7%、82.9%;麝香草酚处理导致核盘菌菌丝畸形、皱缩、细胞死亡,可能与菌丝细胞膜损伤有关,表现为相对电导率和丙二醛含量上升;麝香草酚还可诱导菌丝细胞甘油含量显著上升,发生渗透胁迫;50μg/mL麝香草酚处理青菜可显著抑制核盘菌在叶片上的侵染;在接种核盘菌的青菜叶片中,麝香草酚诱导特异型抗性基因BrMPK3和BrMPK4的表达量上调,并显著提升青菜品质(包括叶绿素、蛋白质、可溶性糖、维生素C含量提升)。麝香草酚可有效控制核盘菌对青菜的侵染,表现为抑菌效应和诱导寄主抗性效应。

羊肚菌菌核形成影响因素与分子机制研究进展

总结了羊肚菌(Morchella spp.)菌核的重要性、形成过程、交配型,影响羊肚菌菌核形成的营养、环境、共栖生物、种或菌株、胞外酶活性等因素及菌核形成的分子机制,并展望未来的研究方向,旨在为羊肚菌菌核形成机制的深入研究提供参考。

实验室自繁大豆菌核萌发影响因素研究

采用人工繁育菌核进行萌发试验,研究人工繁育菌核与自然菌核萌发区别及培养时间、形成菌核的菌龄和保存方式对菌核萌发的影响。结果表明,PDA平板培养菌核萌发时间较少,采集菌核萌发时间最长及萌发率最低;PDA平板15 d形成菌核萌发时间短,培养30和45 d菌核萌发时间无明显差异;不同菌龄形成菌核萌发无差异;繁育菌核直接培养萌发时间最短,干燥储存时间越长,后续萌发培养时间越长。

羊肚菌菌核生物发生及其影响因素研究进展

菌核是由真菌营养菌丝体交织聚集形成的一种休眠结构,可以抵御逆境,提高存活率。形成菌核是羊肚菌(Morchella)生命周期中的一个重要阶段,但其形成机理、影响因素以及与产子实体之间的关系尚不明确,阻碍了羊肚菌人工栽培技术的发展。随着羊肚菌田间栽培的推进,包括其菌核发育在内的多学科基础生物学研究越来越受到重视。本文对形成菌核的主要真菌种类、羊肚菌菌核多样性和发育,以及影响羊肚菌菌核形成的因素进行综述,以期为羊肚菌研究者和栽培技术人员提供参考,进而推动羊肚菌产业的持续和稳定发展。

防控花生白绢病的根际放线菌分离鉴定及防效评价

花生白绢病是由齐整小核菌Sclerotium rolfsii Sacc.引起的发生在花生茎基部的真菌病害,严重制约花生的品质与产量。本研究利用稀释涂布法从怀牛膝根际土壤中分离纯化了116株放线菌,并从中筛选鉴定了能够防控花生白绢病的菌株。通过平板对峙试验筛选得到两株抑菌活性较好且稳定的菌株Soil-1-5和Soil-3-28,它们对齐整小核菌的抑制率分别为80.43%和92.34%。根据形态学观察、生理生化试验及16S rRNA基因序列分析鉴定,两株菌分别被鉴定为疮痂链霉菌Streptomyces scabiei和藤黄灰链霉菌Streptomyces luteogriseus。采用菌丝生长速率法测定了拮抗菌株无菌发酵滤液对植物病原菌的抑制作用。结果表明,菌株Soil-1-5和Soil-3-28的无菌发酵滤液稀释5倍后对齐整小核菌的抑制率分别为73.67%和57.11%,且对禾谷镰孢菌等6种植物病原菌均有不同程度的抑制作用。此外,两菌株的无菌发酵滤液对齐整小核菌菌核萌发和菌核形成也有较好拮抗作用。盆栽试验结果表明,菌株Soil-1-5和Soil-3-28对花生白绢病的防治效果分别为51.92%和31.74%,其中菌株Soil-3-28对花生生长有促进作用。综上,菌株Soil-1-5和Soil-3-28对花生白绢病有较好的防治效果,具有潜在应用价值。

花生白绢病菌拮抗细菌的分离鉴定及其温室抑菌促生效果

由齐整小核菌Sclerotium rolfsii Sacc.侵染引起的土传真菌性病害——花生白绢病,严重威胁花生的生产。为挖掘对花生白绢病有较好防治效果的拮抗细菌,利用稀释涂布法从感染S.rolfsii的植株根际土壤中分离得到570株细菌,采用平板对峙法筛选出32株S.rolfsii拮抗细菌,结合16S rRNA基因序列、形态学及生理生化特征,确定细菌的分类学地位。通过S.rolfsii与拮抗细菌的发酵液共培养和盆栽试验结果表明:在所筛选的32株拮抗细菌中,4株细菌HNZZ23、HNKF5、HNXX14和HNXX169对S.rolfsii的抑制时间长,抑制效果好,其发酵液对S.rolfsii菌丝生长的抑制率分别为85.30%、85.88%、85.16%和91.00%,对S.rolfsii菌核萌发的最大抑制率分别为100%、85.33%、82.67%和93.33%。菌株HNZZ23和HNXX14的发酵液对盆栽花生白绢病的防治效果分别为30.50%和25.73%,且菌株HNZZ23对花生植株的生长具有显著的促进作用。经分类学鉴定,菌株HNZZ23、HNKF5、HNXX14和HNXX169分别为萎缩芽孢杆菌Bacillus atrophaeuse、短小芽孢杆菌B.pumilus、铜绿假单胞菌Pseudomonas aeruginosa和枯草芽孢杆菌B.subtilis。本研究结果表明,感病花生根际细菌HNZZ23、HNKF5、HNXX14和HNXX169对花生白绢病原菌S.rolfsii具有良好的拮抗作用和生防潜力,可为花生白绢病的生物防治提供优质菌种资源。

贝莱斯芽孢杆菌HYN-9的分离、鉴定及其对花生白绢病的生防作用

本研究旨在分离和鉴定对花生白绢病菌(Sclerotium rolfsii Sacc.)具有高效拮抗作用的细菌菌株。采用稀释涂布平板法从废弃的黄芩药渣中分离到18株细菌,其中菌株HYN-9对S.rolfsii的抑菌活性最强,抑菌带宽度为10.92 mm。基于形态特征、生理生化特征和16S rDNA序列,将菌株HYN-9鉴定为贝莱斯芽孢杆菌(Bacillus velezensis)。菌株HYN-9的无菌发酵滤液对S.rolfsii菌丝生长的抑制率为70.01%,并对菌核的形成和萌发也均有较强的抑制作用。此外还发现HYN-9产生的挥发性气体能明显抑制S.rolfsii的生长,抑菌率达88.79%。盆栽试验表明拮抗细菌HYN-9能够明显降低花生白绢病的发病率和感病指数,对花生白绢病的防效为73.23%,显著高于接种多菌灵药剂47.62%的防效(P<0.05)。拮抗细菌HYN-9也能提高花生防御酶活性,接种HYN-9后花生叶片的苯丙氨酸酶(PAL)、多酚氧化酶(PPO)和过氧化物酶(POD)活性显著增加,进而增强花生对病害的系统抗性。以上结果表明贝莱斯芽孢杆菌HYN-9对S.rolfsii具有较强的拮抗作用,是一株具有开发应用潜力的花生白绢病生防菌株。

栽培羊肚菌培养菌丝生长的形态特征

选取从西宁及周边地区栽培种植户采集的来源不同的10个羊肚菌栽培菌株(其中5个六妹羊肚菌Morchella sextelata、3个梯棱羊肚菌M.importuna和2个七妹羊肚菌M.eximia)进行18℃避光马铃薯葡萄糖琼脂培养基(Potato Dextrose Agar Medium)培养,并于长3 d、8 d、14 d分别进行拍照观察和统计分析。通过3D合成图像观察,将菌核(sclerotium)生长划分为五个阶段,分别为:(1)少量菌丝聚集缠绕;(2)大量菌丝聚集成菌丝团;(3)菌核边缘明显;(4)菌核密结生长;(5)菌核发育成熟。观察到菌丝融合,主要表现为:菌丝间相互接触并融合成网状、棒状;观察到厚垣孢子(Chlamydospore):前期为无色或棕黄色,后期变为红棕色覆于菌核上;孢子囊果形态:黄色肉质光滑球状体。实验表明,不同菌种在菌丝生长、菌丝融合、菌核发育以及厚垣孢子发育、生长时间上有差异,实验观察到的:3D成像图片和测量的数据,为羊肚菌全态性(Holomorph)认识提供资料积累。

不同松树树种段木栽培茯苓产量对林分郁闭度的响应

【目的】探明林分郁闭度和不同松树树种段木组合栽培对茯苓产量的影响,为提高茯苓栽培产量提供科学依据。【方法】以茯苓为研究对象,选择低、中、高3种郁闭度林分,分别采用湿地松、马尾松和火炬松3种松树段木栽培茯苓,考察松树不同松种与林分郁闭度组合处理茯苓菌核个数、单个鲜重和总鲜重,通过方差分析和多重比较筛选树种与林分郁闭度的最佳搭配方案。【结果】湿地松段木栽培茯苓水溶性多糖含量为0.49%,马尾松和火炬松段木栽培均为0.26%。湿地松和马尾松段木栽培,中郁闭度林分茯苓单个鲜重和总鲜重显著高于低郁闭度;火炬松段木栽培,高郁闭度林分茯苓总鲜重低于中郁闭度且显著低于低郁闭度。【结论】湿地松、马尾松和火炬松3种松树段木栽培的茯苓综合品质良好,其中,以湿地松段木栽培茯苓的可溶性多糖含量最高。3种郁闭度林分环境下最佳段木栽培搭配方式:低郁闭度林分+火炬松段木,中/高郁闭度林分+湿地松或马尾松段木。

油茶白绢病的发生及防治措施

通过对油茶白绢病的系统调查和分析,揭示其病原生物学特性、发生规律、危害机理及症状,并在此基础上,探讨有效的防治措施,以期通过综合运用农业防治、生物防治以及化学防治等多种手段,为该病害的科学防控及油茶优质高产栽培提供理论依据和实践指导。

Separation of scleroglucan and cell biomass from Sclerotium glucanicum grown in an inexpensive, by-product based medium

Scleroglucan is an extracellular polysaccharide produced by the fungi Sclerotium.Scleroglucan is stable over a broad range of temperatures,pH and salt concentration,thus having great potential in many diverse applications.Common media for scleroglucan production contain expensive components,such as yeast extract,that make the medium cost-ineffective for some industrial applications.Thus we developed a medium which uses Condensed Corn Solubles(CCS),a nutrient-rich byproduct of corn-based ethanol production,to replace expensive components.Methods typically used to recover scleroglucan are also expensive,and can limit commercialization.To evaluate alternative processes for scleroglucan recovery,we prepared scleroglucan in a modified version of Wang’s medium and a CCS-glucose medium.Broth samples were initially subjected to the standard recovery method to develop a complete mass balance,and then we evaluated various treatments to improve scleroglucan recovery.These included heat treatments to lyse cells,using different alcohol precipitants,freezing or refrigerating before recovery,and diluting broth to enhance cell separation.The CCS medium produced 14.2 g/L scleroglucan,compared to 10.1 g/L in the modified Wang’s medium.Based on the standard recovery protocol,we determined that 96%of the scleroglucan was recovered from the initial centrifugation and precipitation.Washing the cell pellet with water and recentrifuging only recovered a minimal amount of scleroglucan,and thus it could be eliminated from protocol to save energy and costs.Lysing cells by boiling or autoclaving did not release more scleroglucan than un-heated samples,and we also found no statistical difference between ethanol,isopopanol,and methanol as scleroglucan precipitants.Refrigerating the broth prior to scleroglucan recovery had no significant effect,while freezing actually decreased scleroglucan recovery.Initially diluting the broth by 0.50 or 0.34 resulted in the greatest scleroglucan recovery,while higher or lower dilutions decreased recovery.The optimum protocol for scleroglucan recovery was a 0.50 dilution of broth prior to centrifugation,no washing of the cell pellet,and use of the least expensive alcohol to precipitate scleroglucan from the supernatant.