Saccharides of ectomycorrhizal fungal sclerotia as sources of forest soil polysaccharides

ABSTRACT

The neutral monosaccharide composition of forest soils differs from that of non-forest soils suggesting there is an accumulation of microbial saccharides. Ectomycorrhizal (ECM) fungi can be responsible as the fungi are typical in forest soils. We investigated neutral saccharides of ECM fungal sclerotia to determine what part it might play in the origin of forest soil polysaccarides. Sclerotial grain (SG) was collected from the O, A1 and A2 horizons of a soil of subalpine forest of Mt. Ontake, central Japan. Neutral saccharides in soil and SG were analyzed by two step hydrolysis with sulfuric acid and gas-chromatography of alditol acetate derivatives. Saccharides accounted for 6.0?16% of the SG by carbon content. The SG contained predominantly easily hydrolysable (EH)-glucose, which accounted for 75–85% of the composition depending on grain size and the soil horizon, followed by mannose (7.7?15%), galactose (2.2?4.8%) and non-easily hydrolysable (NEH)-glucose (1.7?6.1%). The SG contained all of these sugars irrespective of its size. The SG collected from the A1 and A2 horizons contained all sugar components found in that from the O horizon, except for fucose in that from A2 horizon. The monosaccharide composition of SG indicates that accumulation of ECM fungal sclerotial polysaccharides might have been responsible for enlarging the molar ratios of (galactose + mannose) /(arabinose + xylose) and EH-glucose/NEH-glucose of forest soils. The proportions of SG saccharides relative to soil saccharides were 3.6, 1.2, and 0.83% for the O, A1 and A2 horizons, respectively. These levels of the proportion are considerable as ECM fugal sclerotia are the products of a limited species among hundreds and thousands of microbial species inhabiting forest soils. The sclerotia forming ECM fungal species such as Cenococcum geophilum may be key sources of forest soil polysaccharides.     

油菜菌核病菌秋季与春季萌发菌株生物学特性的比较

通过室内和田间试验,观察并比较采自湖北省咸宁市核盘菌秋季萌发菌株与春季萌发菌株的生物学特性。结果表明:9个秋季萌发菌株(aSs-1至aSs-9)和9个春季萌发菌株(sSs-1至sSs-9)在菌丝生长速率、菌核产量、菌核大小和对油菜的致病力等方面均无显著差异(P0.05);室内和田间菌核的萌发均受菌核形成温度和诱导菌核萌发温度的影响,高温形成的菌核易于萌发;核盘菌秋季萌发菌株和春季萌发菌株在菌核萌发特性方面存在明显差异。     

链孢粘帚霉寄生核盘菌菌核差异表达基因的筛选及分析

为克隆和研究链孢粘帚霉Gliocladium catenulatum寄生核盘菌菌核的相关基因,应用抑制消减杂交技术构建了cDNA消减文库并进行了筛选。通过PCR技术从文库中共筛选到1315个阳性克隆,克隆中插入片段大小主要集中于300～600bp之间。随机挑取120个克隆,经测序和同源性分析,获得60条有效序列,其中部分序列所编码的血红素加氧酶、核糖体蛋白L11、细胞色素P450及热激蛋白等均参与机体对胁迫条件的应答反应。11条序列在NCBI数据库中未找到显著匹配的序列,可能为新基因片段。分别将寄生于核盘菌菌核上的粘帚霉cDNA和粘帚霉与核盘菌纯培养的cDNA混合物经RasⅠ酶切后进行标记作为探针,利用反向Northern杂交技术验证了所选取的25条序列全部为差异表达基因片段。     

Biological control of Rhizoctonia solani on potatoes by antagonists. 2. Sprout protection against soil-borne R. solani through seed inoculation with Verticilium biguttatum

Verticillium biguttatum isolate M73, when inoculated on the seed tuber, was found to protect young potato sprouts againstRhizoctonia infection from the soil and also, as was demonstrated before, against infection from sclerotia on seed tubers. The experiments were performed under laboratory conditions and lasted a few weeks. V. biguttatum colonizes the surface of the sprouts and may be regarded as a natural inhabitant of the surface of sprouts and presumably also of stems and stolons in a later stage. This fungus may be of value for biological control ofR. solani.Samenvatting Isolaat M73 van de schimmelVerticillium biguttatum, indien geënt op de pootknol, blijkt de aantasting van aardappelspruiten doorRhizoctonia solani te kunnen verminderen. Zowel de aantasting vanuit de grond en, zoals reeds eerder aangetoond, vanaf sclerotiën op de pootknol worden minder. Verticillium biguttatum koloniseert het oppervlak van de spruiten en kan beschouwd worden als een natuurlijke bewoner van het oppervlak van spruiten en wellicht ook van stengels en stolonen in een later stadium. In kort durende proeven, als hier beschreven, lijktV. biguttatum goed bruikbaar voor een biologische bestrijding vanR. solani.     

Survival of Botrytis Cinerea in Southeastern Spanish Greenhouses

The relative importance of sclerotia and mycelia of Botrytis cinerea Pers ex Fr. as structures of survival in southeastern Spanish greenhouses was investigated. Sclerotia were not found in the SE region, neither on plant debris nor on living plant material, suggesting it may serve only a minor role in epidemic development. B. cinerea survived mostly as mycelium. The percentage of artificially inoculated tomato stem pieces from which mycelium was recovered, was used to quantify its survival rate. Outside the greenhouses, mycelium survived in 33% and 5% of the tomato stem pieces 110 days after inoculation in 1995 and 1997, respectively. After the same number of days inside the greenhouses, no mycelium was recovered from stem pieces in 1995, and in 1997 only 7% of the stem pieces contained mycelium. Survival of mycelium outside and inside the greenhouses was significantly (P < 0.05) different after 47, 83, and 110 days of exposure to field conditions in 1995, but they were not different in 1997. Under controlled conditions, mycelium of B. cinerea lost viability at 100% relative humidity at temperatures ranging from 5 to 40°C, suggesting that air temperature and relative humidity accounted for loss of viability of mycelium.     

Effect of haulm destruction supplemented by cutting off roots on the incidence of black scurf and skin damage,flexibility of harvest period and yield of seed potatoes in field experiments

In field experiments, supplementing chemical haulm destruction (CHD) with cutting off roots resulted in a lower incidence of black scurf and skin damage (ripping off the skin) at harvest date than CHD alone. The lower susceptibility to skin damage at harvest allowed harvesting to beging on an earlier date, when only a few sclerotia ofRhizoctonia solani had developed. Furthermore, black scurf often developed more slowly after haulm destruction if roots had been severed and this enabled harvesting to be postponed.At harvest, gross yield was highest if roots had not been cut through prior to CHD: the extra weight existed merely of water. Weight loss during storage, however, as well as grading losses resulting from black scurf were greater after CHD alone. This resulted in an equal or even lower net yield after CHD alone than after CHD supplemented by root severing. The favourable effects of supplementing CHD with cutting off roots almost equaled those of the mechanical removal, often called haulm pulling or plant pulling. Factors that may affect the development of black scurf are discussed.Samenvatting Pootaardappelen raakten minder bezet met lakschurft en werden minder ontveld bij de oogst na looftrekken of na wortelsnijden plus doodspuiten dan na loofklappen plus spuiten. Al vanaf de derde dag na trekken of wortelsnijden plus doodspuiten was de mate van ontvelling zo gering, dat met het oogsten kon worden begonnen. Op zo'n vroeg tijdstip na loofdoding konden knollen worden geoogst met nog weinig lakschurft, want de stimulering van lakschurft werd pas zichtbaar vanaf 7 à 10 dagen na loofdoding. Lakschurft ontwikkelde zich het traagst na looftrekken. Ook wortelsnijden bij het doodspuiten gaf meestal een tragere ontwikkeling van lakschurft dan klappen plus spuiten. Daardoor konden na wortelsnijden plus doodspuiten en vooral na looftrekken op nog latere tijdstippen knollen met weinig lakschurft worden geoogst.Na klappen plus spuiten was het bruto gewicht bij de oogst het hoogst. Dat meergewicht bleek louter uit water te bestaan en het verloop in knolvochtgehalte leek erop te wijzen dat de wortels nog gedurende een week na doodspuiten blijven functioneren. Dat gaf wel knollen met een hoger vochtgehalte die meer ontvelden en meer vocht en dus gewicht verloren. Dit groter gewichtsverlies bij bewaren en de hogere leesverliezen door lakschurft deden de meeropbrengst teniet: zo werd het netto knolgewicht na klappen plus spuiten, al naar gelang het moment van oogsten, meestal gelijk aan, of lager dan het netto knolgewicht na het trekken of na wortelsnijden plus doospuiten. In dit artikel worden de factoren besproken, die mogelijk van invloed zijn op de ontwikkeling van lakschurft.     

Biological control of Rhizoctonia solani on potatoes by antagonists. 3. Inoculation of seed potatoes with different fungi

Inoculation of seed potatoes withVerticillium biguttatum and three other hyperparasitic fungi, alone or in combination, resulted in statistically significant reduction of infestation of potato plants byRhizoctonia solani. Gliocladium roseum, Trichoderma hamatum andHormiactis fimicola did not show prolonged protection againstR. solani under farming conditions.H. fimicola, however, inhibited mycelial growth ofR. solani in vitro, particularly in the lower temperature range whereV. biguttatum did not show any growth. Combining these two antagonistic fungi may be advantageous as they cover the entire temperature range in whichR. solani is active. At the end of the vegetation period,V. biguttatum was superseded byG. roseum.Production of sclerotia on newly formed tubers from seed potatoes inoculated withV. biguttatum (alone or in combination with the other three antagonists) was significantly reduced.Samenvatting Inoculative van pootaardappelen metVerticillium biguttatum, apart of sament met drie andere hyperparasieten, had een gunstig effect op het onderdrukken vanRhizoctonia solani op de plant;Trichoderma hamatum, Gliocaldium roseum enHormiactis fimicola, ieder apart toegediend, boden de plant op lange termijn geen bescherming tegenR. solani onder praktijkomstandigheden.H. fimicola bleek bij lage temperatuur, waarbijV. biguttatum geen groei-activiteit meer vertoonde, op hyfen en sclerotiën vanR. solani te kunnen groeien. Toepassing van deze schimmel enV. biguttatum in een gemengde inoculatie zou over een breder temperatuurtraject effectief kunnen zijn.Tegen het einde van het groeiseizoen vond er op de ondergrondse plantedelen een verschuiving plaats, waarbijG. roseum meer op de voorgrond trad. Verondersteld wordt dat de afname vanV. biguttatum op stolonen hiervan een gevolg was.De produktie van sclerotia op nieuwe aardappelen afkomstig van metV. biguttatum behandeld pootgoed (alleen of met andere hyperparasitaire schimmels) bleek sterk verminderd te zijn. Vooral dit gegeven maakt de biologische bestrijding vanR. solani interessant:V. biguttatum blijkt ook op lange termijn effectief te zijn.     

Use of sclerotia of<Emphasis Type="Italic">Sclerotinia sclerotiorum</Emphasis> for efficient production of conidia of<Emphasis Type="Italic">Coniothyrium minitans</Emphasis> in liquid culture

A study was conducted to determine the feasibility of using sclerotia ofSclerotinia sclerotiorum for producing conidia ofConiothyrium minitans in liquid culture. The medium (SST) was made of water containing 2.0, 1.5, 1.0 or 0.5% (w/v) ground sclerotia ofS. sclerotiorum and 100 μgl ⁻¹ thiamine hydrochloride (HCl). One ml of conidial suspension (2 × 10⁷ conidia ml⁻¹) ofC. minitans LRC 2534 was inoculated into 100 ml of SST medium or control (thiamine HCl in water) and incubated at 20 ± 2°C on a shaker at 200 rpm. Subsamples were removed periodically and examined under a compound microscope. Conidia in the SST media germinated within 24 h, developed into branched hyphae within 48 h, produced pycnidia after 3–4 days, and the pycnidia released mature conidia after 7 days. Production of conidia varied with the concentration of sclerotia in the SST medium. It was lower (3.6 × 10⁶ conidia ml⁻¹) at 0.5% but higher (1.2 × 10⁸ conidia ml⁻¹) at 2%. The new conidia were viable and the colonies developing from them showed the original morphological characteristics. It was concluded that using SST liquid medium as a substrate for mass production of conidia ofC. minitans has potential for use in commercial development of this mycoparasite as a biocontrol product. http:www.phytoparasitica.org posting Jan. 23, 2007.     

向日葵菌核病菌核秋季田间生成量的估算

通过田间调查及室内分析,从198个病盘分一、二、三、四级分别建立四个指数方程。可作为估算秋季田间病盘上菌核生成量的依据。