Biological diversity of Rhizoctonia solani (AG-3) in a northern potato-cultivation environment in Finland

A total of 119 isolates of Rhizoctonia were collected from stem canker lesions, stolon and root lesions, hymenia on stems, or from black scurf on tubers of potato plants ( Solanum tuberosum ) in Finland (latitudes 60–67°N). All isolates except three belonged to anastomosis group 3 (AG-3) of R. solani , as determined by phylogenetic analysis of the internal transcribed spacer sequences (ITS1 and ITS2) of ribosomal RNA (rRNA) genes. Sensitivity of the 119 isolates to the fungicide flutolanil was tested in vitro (EC₅₀ values 0·14–0·75  µ g active ingredient mL⁻¹). The isolates also varied considerably in growth rate (5·1–14·8 mm day⁻¹). The severity of disease caused by 99 isolates was determined based on the proportion of potato sprouts affected by lesions, discoloration or death, which was c . 1–60%. Only two isolates that were able to cause severe symptoms showed particularly low sensitivity to the fungicide and rapid growth rate. One isolate each of anastomosis groups AG-2-1 and AG-5 and an unknown, binucleate Rhizoctonia sp. were detected. The AG-5 isolate and the binucleate isolate caused mild symptoms on potato sprouts, whereas the AG-2-1 isolate was not pathogenic. Taken together, AG-3 of R. solani was the predominant causal agent of the stem canker and black scurf diseases of potato in Finland and showed considerable variability in disease severity, fungicide sensitivity and growth rate in vitro .     

Mycoparasitism of Helminthosporium solani by Acremonium strictum

ABSTRACT Isolates of Helminthosporium solani, the causal agent of silver scurf of potato, collected from multiple locations consistently show white sectoring and rings, differential coloration, and reduced sporulation in culture. It has been accepted that this growth pattern is normal for H. solani cultures. Scanning electron microscopy confirmed the presence of a contaminating fungus in close association with cultures of H. solani. Repeated hyphal tip isolation techniques were used to separate H. solani from the fungal contaminant. Resultant pure cultures of H. solani were uniformly black in color, without white sectors or rings. The contaminating fungus was identified as Acremonium strictum. The purpose of this study was to elucidate the relationship between A. strictum and H. solani, and evaluate the impact of the fungicolous A. strictum on the growth and biology of H. solani. In vitro studies demonstrated that A. strictum significantly reduced sporulation of H. solani isolates from 65 to 35%, spore germination from 53 to 43%, and mycelial growth from 40 to 32% compared with noncontaminated cultures of H. solani. These data indicate that A. strictum is antagonistic to H. solani, and can be considered a mycoparasite. A. strictum reduced H. solani conidia production on minitubers, thereby reducing inoculum for infection. However, treatment with A. strictum does not reduce silver scurf of previously infected tubers. Further studies are warranted to determine the full potential of A. strictum as a biological control agent of H. solanii-incited silver scurf of stored potato tubers and the most effective manner of use.     

Relationships between stem canker, stolon canker, black scurf (Rhizoctonia solani) and yield of potato (Solanum tuberosum) under different agronomic conditions

Relationships between diseases caused by Rhizoctonia solani on different parts of potato plants ( Solanum tuberosum ) at different stages of crop growth were investigated under selected agronomic conditions. The effects of different densities of tuber-borne inoculum, date of planting, irrigation, size of seed tubers and their interactions on the incidence of stem and stolon canker during crop growth, the incidence and severity of black scurf and the yield of progeny tubers at harvest were quantified in a multifactorial experiment. Differences in the incidence of stem canker, stolon canker and black scurf were dominated by the effect of density of inoculum on seed tubers at planting. Highly positive correlations between the disease variables indicated a close relationship between the incidence of disease at each stage of crop growth although the degree of association between variables measured at an early growth stage and those measured at progressively later stages of crop growth weakened as the time interval increased. Total yield of progeny tubers was not affected by the density of tuber-borne inoculum although there was a shift in the size distribution, with a decrease in the yield of main-sized tubers and an increase in the yield of baker- and oversized tubers at the higher density of inoculum. Of the remaining factors, the effect of season tended to be more pronounced than any of the agronomic treatments although the use of irrigation and later dates of planting did influence the incidence of infection to a limited extent.     

Novel in vivo use of a polyvalent Streptomyces phage to disinfest Streptomyces scabies-infected seed potatoes

A highly virulent and polyvalent Streptomyces phage was isolated from a potato field near Albany, Western Australia. The efficacy of the isolated phage to disinfest seed potato tubers artificially inoculated with a common scab-causing streptomycete was evaluated. The phage suspension was prepared in a mini-bioreactor. Diseased potatoes were bathed in a phage suspension (1 × 10⁹ plaque-forming units per mL) for 24 h. The suspension was constantly circulated within a novel 25 L phage bath by means of an air-sparging pipe driven from an air compressor. Phage-treated scab-affected seed potatoes planted into free-draining polystyrene boxes containing steam-pasteurized field soil produced tuber progeny with significantly ( P  < 0·05) reduced levels of surface lesions of scab (1·2%) compared with tubers harvested from nonphage-treated tubers (23%). The number of scab lesions was also significantly reduced ( P  < 0·05) by phage treatment of mother tubers. No significant differences were recorded in weight, size or number of harvested tubers from phage-treated or nontreated mother tubers. This is the first in vivo study that has used Streptomyces phage to significantly disinfest seed potatoes of Streptomyces scabies and thereby reduce contamination of soil from seed-tuber-borne inoculum and reduce infection of daughter tubers.     

Development of resistance to thiabendazole in Helminthosporium solani (silver scurf) as a result of potato seed tuber treatment

Applying thiabendazole to potato seed tubers affected with silver scurf caused by Helminthosporium solani sensitive to thiabendazole decreased the severity of disease on progeny tubers at harvest, but about 50% of the isolates from these were resistant to the fungicide. The disease was not decreased when samples of the progeny tubers were treated with thiabendazole and planted in the following year, and the incidence of resistant isolates increased. Resistant isolates continued to be present when tubers were planted in the next 2 years without fungicide treatment. Treatment with a mixture of thiabendazole and imazalil also decreased the disease and fewer isolates were resistant than when treated with thiabendazole alone, although the proportion increased after treatment with the mixture in the following year. When seed tubers were infected with thiabendazole-resistant H. solani , silver scurf on progeny tubers was not affected by thiabendazole applied to the seed tubers but was decreased by the mixture of thiabendazole and imazalil. Imazalil was equally effective against H. solani sensitive or resistant to thiabendazole.
Some isolates of H. solani had grey aerial mycelium and of 516 of these isolates obtained in 4 years 29% were resistant to the fungicide. Other isolates produced small, black colonies and their frequency increased with thiabendazole treatment of seed tubers. Of 244 of these isolates, 62% were resistant.     

木霉对草坪褐斑病的拮抗效果及耐药性

通过木霉属5菌株与大连高尔夫球场上草坪褐斑病菌的对峙培养试验,研究结果表明:哈茨木霉Thar1菌株、哈茨木霉Thar2菌株、深绿木霉Tat菌株、钩状木霉Tha菌株及桔绿木霉Tci菌株对病原菌的抑制率为:66%、73%、71%、77%、55%,而相对抑菌效果分别为2.06、3.94、2.35、3.54、2.27,可以作为草坪褐斑病菌的生防菌加以利用。在这5株木霉中以哈茨木霉Thar2对草坪褐斑病菌的拮抗作用最强。观察结果表明,哈茨木霉Thar2对草坪褐斑病菌的拮抗机制主要表现为生长竞争、重寄生及产生抗菌物质。     

Integrated control of Rhizoctonia solani by methyl bromide and Trichoderma harzianum

Under laboratory conditions, isolate TH–203 of Trichoderma harzianum was found to be tolerant of up to 20 000 ppm methyl bromide (MB) (v/v), whereas the plant pathogen Rhizoctonia solani was susceptible to a dose of less than 9000 ppm (v/v). Exposure to sub–lethal concentrations of MB had no effect on the in vitro antagonistic ability of T. harzianum . Soil fumigation with MB at the equivalent of a commercial dose of 500 kg/ha did not reduce the population of Trichoderma in soil and allowed rapid colonization of Trichoderma to develop in the soil.
Under greenhouse conditions a combination of T. harzianum and a reduced dose of MB (equivalent to 200 kg/ha) completely controlled disease incidence of R. solani in bean seedlings compared with controls in untreated soils. Similar disease control was achieved with the recommended dose of MB. Under field conditions, the combination of 200 kg/ha MB and T. harzianum gave a significant synergistic effect on damping–off of carrot seedlings caused by R. solani , and had a similar effect on growth, yield and disease control to that of the recommended dose.
T. harzianum was also able to prevent reinfestation by R. solani in fumigated soils.     

碳酸氢盐对马铃薯黑痣病菌毒力及田间防效

为明确碳酸氢盐对马铃薯黑痣病菌的毒力及田间防效,采用菌丝生长速率法,测定了碳酸氢盐对马铃薯黑痣病菌的毒力;并通过盆栽防治试验及田间防治试验,研究了碳酸氢盐对马铃薯黑痣病的防治效果。结果表明:碳酸氢铵抑制马铃薯黑痣病菌菌丝生长的有效中浓度EC50仅为0.71g/L,碳酸氢钾EC50为3.8g/L;在PSA培养基中同时加入0.79g/L碳酸氢铵、1g/L碳酸氢钾与在PSA培养基中含1.58g/L碳酸氢铵的抑菌效果一样;尿素对马铃薯黑痣病没有防治效果。碳酸氢铵与碳酸氢钾混配对该病有良好的防治作用,相对尿素最高防治效果可达到63.4%。覆膜栽培能够提高碳酸氢盐的防效和马铃薯产量。碳酸氢盐可用于田间马铃薯黑痣病的防治。     

Heat treatment of seed tubers for control of potato blackleg (Erwinia carotovora subsp. atroseptica) and other diseases

The thermal death points of Erwinia carotovora subsp. atroseptica and subsp. carotovora were determined in relation to duration of heat treatment, age of culture and culture medium. No isolates cultured in liquid media survived heating at 53°C for 5 min while those on solid media were killed by heating at 54°C for 10 min. After immersing naturally contaminated potato tubers for 10 min in water at 55°C, Erwinia could not be detected. The same treatment of naturally or artificially contaminated seed tubers gave complete absence of blackleg infection in the field and decreased the amounts of powdery scah(Spongospora subterranea) and black scurf (Rhizoctonia solani) on progeny tubers.     

Infection of potato by Rhizoctonia solani: effect of anastomosis group

Glasshouse and field experiments showed that the pathogenicity and disease type on potato varied between different anastomosis groups (AGs) of Rhizoctonia solani. For example, severe stem and stolon disease developed in plants inoculated with a single isolate of AG3PT and AG5. Severe root disease was observed with single isolates of AG8 and to a lesser extent AG3PT, but rarely with single isolates of the other AGs tested. In both field and glasshouse experiments the AG2‐1 isolate (X81) produced only small lesions (<5 mm). However, this was not representative of two other AG2‐1 isolates. When AG2‐1 isolates of the three different rDNA IGS1 types were tested in a glasshouse trial, one caused more severe stem and stolon infection than AG3PT. In the field experiment, the yield of tubers, by weight, was significantly less (P < 0·05) in all inoculated plants than for uninoculated (control) plants. Yield losses were greatest and tuber numbers smallest in plots inoculated with an AG8 isolate, suggesting that root infection is important in determining quantitative yield loss. The incidence of black scurf was greatest in the progeny tubers in plots inoculated with AG3PT (83·9%), whereas only very small amounts of black scurf developed on tubers from plants infected with AG2‐1 (510 bp) or AG5 isolates. This is supported by laboratory tests, where isolates of AG3PT produced significantly more sclerotia on potato dextrose agar than isolates of AGs 2‐1, 4, 5 and 8.     

Anastomosis group and pathogenicity of isolates of Rhizoctonia solani from potato crops in South Australia

Isolates of Rhizoctonia collected from the stems, roots, tuber sclerotia and soil of potato crops in Virginia and Lenswood, South Australia, were identified to anastomosis groups (AG). Of the 301 multinucleate isolates of Rhizoctonia solani tested, 90% were AG-3, 7% were AG-4 and 2% were AG-5; 12 isolates were binucleate Rhizoctonia spp. This is the first report of isolates of AG-4 and AG-5 causing disease in potato crops in South Australia. All AG-3, AG-4 and AG-5 isolates tested caused rhizoctonia disease symptoms on the potato cultivar Coliban in pathogenicity trials conducted under glasshotise conditions. Both AG-3 and AG-5 isolates caused black scurf and stem cankers, although symptoms of black scurf were less severe with AG-5. AG-4 isolates produced the most severe stem and stolon cankers of all isolates tested. The pathogenicity of tuber-borne inoculum was confirmed by growing plants from sclerotia-infested tubers. AG-8 isolates from diseased barley and wheat produced severe root cankers and caused loss of feeder roots on inoculated potato plants. Results suggest that rhizoctonia disease in potato fields in South Australia is caused by a combination of different anastomosis groups and this has important implications for crop rotations.     

Effect of inoculum source on root and tuber infection by potato blemish disease fungi

The incidence of potato pathogens on healthy roots of micropropagated (MP) and seed tuber (ST) plants was examined on successive dates during the growing season in two field experiments. Microplants were grown in a glasshouse for 4–5 weeks in perlite or peal-based substrates, and exposed or not to natural inoculum before planting in the field. The seed tubers originated from stocks of visually clean or moderately blemished tubers and were surface-sterilized or not before planting. Polyscytalum pustulans and Helminthosporium solani only infected roots of ST plants and inoculated MP plants. The incidence of P. pustulans was affected by seed tuber-borne inoculum and, in I year, by the substrate. H. solani was detected infrequently on roots. Rhizoctonia solani was present at low frequencies in most root samples, and more ST than MP plant roots were colonized; there were no substrate effects. In 1 year, increased inoculum levels increased root infection, but only in MP roots. Colletotrichum coccodes occurred at high frequencies and was most common in roots of ST plants. Progeny tubers showed some treatment effects when tested in September and after storage for 6 months, but there were no consistent relationships between root and progeny tuber infection.     

Detection of Colletotrichum coccodes from soil and potato tubers by conventional and quantitative real-time PCR

Colletotrichum coccodes is the causal agent of the potato blemish disease black dot. Two PCR primer sets were designed to sequences of the ribosomal internal transcribed spacer (ITS1 and ITS2) regions for use in a nested PCR. The genus-specific outer primers (Cc1F1/Cc2R1) were designed to regions common to Colletotrichum spp., and the species-specific nested primers (Cc1NF1/Cc2NR1) were designed to sequences unique to C . coccodes . The primer sets amplified single products of 447 bp (Cc1F1/Cc2R1) and 349 bp (Cc1NF1/Cc2NR1) with DNA extracted from 33 European and North American isolates of C. coccodes. The specificity of primers Cc1NF1/Cc2NR1 was confirmed by the absence of amplified product with DNA of other species representing the six phylogenetic groups of the genus Colletotrichum and 46 other eukaryotic and prokaryotic plant pathogenic species. A rapid procedure for the direct extraction of DNA from soil and potato tubers was used to verify the PCR assay for detecting C. coccodes in environmental samples. The limit of sensitivity of PCR for the specific detection of C. coccodes when inoculum was added to soils was 3·0 spores per g, or the equivalent of 0·06 microsclerotia per g soil, the lowest level of inoculum tested. Colletotrichum coccodes was also detected by PCR in naturally infested soil and from both potato peel and peel extract from infected and apparently healthy tubers. Specific primers and a TaqMan fluorogenic probe were designed to perform quantitative real-time (TaqMan) PCR to obtain the same levels of sensitivity for detection of C. coccodes in soil and tubers during a first-round PCR as with conventional nested PCR and gel electrophoresis. This rapid and quantitative PCR diagnostic assay allows an accurate estimation of tuber and soil contamination by C. coccodes .     

Influence of abiotic factors,inoculum source,and cultivar susceptibility on the potato tuber blemish diseases black dot (Colletotrichum coccodes) and silver scurf (Helminthosporium solani)

Black dot and silver scurf are potato blemish diseases whose economic impact has increased in recent years. Because their symptomatology on tubers is visually similar, disease assessment does not usually differentiate between the two pathogens, which share the same ecological niche. The epidemiology of black dot has been extensively studied, especially in the UK, but the factors that influence silver scurf have been less investigated. In this study, the influence of cultivar, source of inoculum, and environmental conditions on both diseases was studied in field trials over a three-year period (2016–2018) in Switzerland. Planting minitubers did not prevent either disease in daughter tubers, indicating the contribution of soil as an inoculum reservoir. An arbitrary threshold of Colletotrichum coccodes soil inoculum could be set to discriminate between low and high disease risk. For the first time, Helminthosporium solani DNA was detected in stolons, and infections appeared earlier in stolons than in tubers. H. solani stolon and tuber infections usually appeared later in the season than those of C. coccodes. Black dot severity correlated positively with precipitation, while silver scurf severity correlated positively with temperature. Table potato cultivars commonly grown in Switzerland exhibited significant differences in susceptibility to both diseases, and cultivars with low susceptibility to both silver scurf and black dot were identified. These results gave new insights into understanding the factors driving the epidemiology of potato blemish diseases and may contribute to building a risk assessment scheme to manage both diseases simultaneously.     

Detection of Colletotrichum coccodes and Helminthosporium solani in soils by bioassay

The sensitivity of a bioassay in detecting soil inoculum of Colletotrichum coccodes and Helminthosporium solani was examined using potato minitubers and microplants. Tests were conducted on soils which were collected from fields in which the interval after a previous potato crop differed, and which were also artificially infested with conidia or microsclerotia. For C. coccodes , determining plant infection based on the occurrence of infected roots after 9–12 weeks was a sensitive method for detecting and quantifying the amount of inoculum in soil. Infestations of less than 0·4 microsclerotia per g soil were detected in artificially infested soils. A semiselective medium, developed for isolating C. gloeosporioides from pepper, detected soil infestations by C. coccodes as low as nine conidia or one microsclerotium per g soil in artificially infested soil. For H. solani , infection on minitubers was a sensitive measure, with soil inoculum of fewer than 10 conidia per g soil being detected. Soil infestation could be quantified by assessing the percentage surface area of minitubers covered by sporulating lesions, which was strongly related to the amount of soil infestation. The results of these bioassay tests were compared with published results for real-time quantitative PCR assays on the same soils. The two methods were in good agreement in artificially infested soils, but the bioassay appeared to be more sensitive with naturally infested soils.     

农杆菌介导的哈茨木霉T-DNA转化系统优化及拮抗能力突变子的性质分析

采用单因子试验方法研究了农杆菌EHA105介导的哈茨木霉Th-33转化过程中,各主要因素对转化效率的影响,建立了高效的转化系统,使农杆菌转化哈茨木霉的效率达到60～150个转化子/10^6个木霉孢子,利用该转化系统构建了含有8000多个转化子的T-DNA插入突变库。通过转化子与立枯丝核菌的对峙试验,从1260株转化子中筛选到23株拮抗能力发生变化的突变子。随机挑选5株突变子对其遗传稳定性进行分析,表明5株突变子都具有稳定性,聚合酶链式反应（PCR）表明上述突变子均有T-DNA片段的插入。     

一株拮抗4种马铃薯病原真菌的拟康宁木霉的分离鉴定

本研究从黑龙江省土壤中分离获得一株木霉, 运用生物学特征和ITS序列分析相结合的方法将其鉴定为拟康宁木霉Trichoderma koningiopsis?薯块活体抑菌测定表明, 该木霉对接骨木镰孢Fusarium sambucinum?马铃薯早疫病菌Alternaria solani?立枯丝核菌Rhizoctonia solani (AG3和AG5)等病原真菌引起的马铃薯病害具有抑制作用?对峙培养试验结果显示, 该木霉对上述病原菌的生长具有拮抗作用, 平均抑制率均超过50%, 且该木霉生长速度快于上述病原真菌, 以竞争生长方式抑制病原菌菌丝生长和菌核形成?采用扫描电子显微镜观察发现, 该木霉可通过缠绕或并行生长等方式寄生在上述病原真菌菌丝上?以上结果表明, 从土壤中分离获得的拟康宁木霉具备良好的生防潜力, 可为马铃薯种植中多种真菌病害的统防统治提供良好的生防菌株?     

Integrated control of Rhizoctonia solani and fusarium solani in tobacco transplants with Trichoderma hurziunum and triadimenol

Trichoderma cultures were isolated locally from soils and screened on potato-dextrose agar against Rhizoctonia solani. Several isolates of T. harzianum reduced the growth and build up of populations of R. solani and to a lesser extent of Fusarium solani in sterilized soil. In field experiments, biological control of R. solani and F. solani infections in tobacco transplants was achieved by adding T. harzianum (T77) to methyl-bromide-fumigated seedbeds before seed was sown. There was also increased growth and leaf yield that was not directly correlated with disease control. Triadimenol fungicide, integrated with the Trichoderma treatment, enhanced disease control.     

Evaluation of Trichoderma harzianum for controlling root rot caused by Phytophthora capsici in pepper plants

The ability of Trichoderma harzianum to control the rotting of pepper ( Capsicum annuum ) plant roots caused by Phytophthora capsici was studied. Interactions between the fungi were assessed in vitro on three culture media (V8c, Czapek and 2% water agar) and in vivo in plants grown in a substrate inoculated with P. capsici and T. harzianum . Studies on mutual antagonism in vitro showed that P. capsici was inhibited by T. harzianum ; however, the intensity of inhibition differed according to the medium used, being greatest on Czapek. Analysis of the fungal populations in the plant growth substrate showed that T. harzianum consistently reduced that of P. capsici over time. This reduction in the pathogen population was associated with a reduction in root rot of between 24 and 76%, although plant growth (dry weight) was still reduced by 21.2–24.7%, compared with the uninoculated control. In the absence of T. harzianum with the same pathogen inoculum levels, the reduction in dry weight was 59.8–68.6%, suggesting that T. harzianum reduced the damage.     

Perspectives of green-crop-harvesting to control soil-borne and storage diseases of seed potatoes

An overview is given on the history of vine killing meant to reduce virus transmission to potato seed tubers. With mechanical harvesting, problems arise with respect to skin damage and pathogens, which strongly develop on the decaying vines and roots, such asRhizoctonia solani, Phoma exigua var.foveata andErwinia spp. Green-crop-harvesting (GCH), a recently developed fully mechanical vine killing method, is marked by vine destruction, lifting tubers, placing them on a soil bed followed by covering them with soil and an in situ healing period of at least ten days in the newly formed ridges.GCH is characterized by a very low level of skin damage, excellent healing conditions and reduced development of above mentioned pathogens.By applying fungicides or antagonistic organisms with the first lifting, effective control was achieved of a number of important potato diseases. Late blight was controlled by fungicides. Control of black scurf and gangrene was improved by the application of either fungicides or antagonists. Erwinia spp. were found to be effectively controlled by antagonists. Perspectives of GCH to control these diseases and other important ones are discussed.