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1.
ABSTRACT A murine hybridoma cell line GD2 secreting an immunoglobulin (Ig)M monoclonal antibody (MAb) was produced against surface antigens from an anastomosis group (AG) 4 isolate of Rhizoctonia solani (teleomorph: Thanatephorus cucumeris). Ascites were produced in mice using GD2 hybridoma cells and used to develop a rapid immunochromatographic lateral flow device (LFD) for the detection of antigens from R. solani and certain related Rhizoctonia spp. The LFD was tested for specificity against surface antigens from related and unrelated soil fungi. Antigens from representative isolates of R. solani AGs 1, 2-1, 2-3, 2-t, 3, 4, 5, 6, 7, 8, 9, 10, 11, and BI gave a positive response in LFD tests, as did antigens from Thanatephorus orchidicola, T. praticola, R. fragariae (teleomorph: Ceratorhiza fragariae), Ceratorhiza goodyerae-repentis, Ceratobasidium cornigerum, and binucleate AGE. Antigens from R. solani AGs 2-2, 2-2IIIB, and 2-2IV and from the related fungi R. carotae, R. cerealis (teleomorph: Ceratobasium cereale), R. crocorum (teleomorph: Helicobasidium brebissonii), R. oryzae (teleomorph Waitea circinata), and R. zeae gave negative responses, as did antigens from a range of unrelated fungi and oomycetes including Fusarium, Gliocladium, Trichoderma, Pythium, and Phytophthora spp. The usefulness of the LFD to detect R. solani was demonstrated in soils naturally infested with R. solani AG3. There was close agreement between results of LFD tests and conventional plate enrichment tests employing selective medium. The specificity of the technique was confirmed by polymerase chain reaction PCR using R. solani AG3-specific primers and by analyses based on sequences of the internal transcribed spacer (ITS)1-5.8S-ITS2 rRNA-encoding regions of unrelated fungi recovered from soil samples. The LFD was used to quantify R. solani AG4 in artificially infested soil samples (chopped potato soil inoculum). Estimates of CFU per gram of soil were derived using a most-probable number technique, which was based on the presence or absence of a detectable signal in the LFD. Estimates of CFU obtained in LFD tests and those obtained in a plate-trapped antigen enzyme-linked immunosorbent assay incorporating MAb GD2 were identical (449 CFU g(-1) of soil).  相似文献   

2.
Mazzola M 《Phytopathology》1997,87(6):582-587
ABSTRACT Rhizoctonia spp. were isolated from the roots of apple trees and associated soil collected in orchards located near Moxee, Quincy, East Wenatchee, and Wenatchee, WA. The anastomosis groups (AGs) of Rhizoctonia spp. isolated from apple were determined by hyphal anastomosis with tester strains on 2% water agar and, where warranted, sequence analysis of the rDNA internal transcribed spacer region and restriction analysis of an amplified fragment from the 28S ribosomal RNA gene were used to corroborate these identifications. The dominant AG of R. solani isolated from the Moxee and East Wenatchee orchards were AG 5 and AG 6, respectively. Binucleate Rhizoctonia spp. were recovered from apple roots at three of four orchards surveyed and included isolates of AG-A, -G, -I, -J, and -Q. In artificial inoculations, isolates of R. solani AG 5 and AG 6 caused extensive root rot and death of 2- to 20-week-old apple transplants, providing evidence that isolates of R. solani AG 6 can be highly virulent and do not merely exist as saprophytes. The effect of binucleate Rhizoctonia spp. on growth of apple seedlings was isolate-dependent and ranged from growth enhancement to severe root rot. R. solani AG 5 and AG 6 were isolated from stunted trees, but not healthy trees, in an orchard near Moxee, WA, that exhibited severe symptoms of apple replant disease, suggesting that R. solani may have a role in this disease complex.  相似文献   

3.
A collection of 241 isolates of Rhizoctonia solani obtained from potato plants grown in different areas in France was characterized for anastomosis grouping, symptomatology on tubers of different cultivars and sensitivity to three fungicides. Most isolates collected belonged to (anastomosis groups (AGs)) AG 3, but 2% and 4% of the isolates were AG 5 and AG 2-1. AG 3 and AG 2-1 isolates were mostly obtained from sclerotia on tubers, but all AG 5, some AG 3 and some AG 2-1 isolates were recovered from superficial tuber alterations, like deformations, corky or scabby lesions. Sclerotia were formed on tubers produced by healthy stem cuttings grown in soil artificially infested with AG 3, but not on tubers grown in soil infested with either AG 5 or AG 2-1. No variation in susceptibility to sclerotial formation was observed among five potato cultivars. In all cases, a large proportion of tubers showed superficial corky lesions, often associated with deformations. The proportion of tubers with lesions and deformations was highest in soil infested with AG 2-1 and significantly lower on cv. Samba in all treatments. All isolates were highly sensitive to flutolanil, iprodione and pencycuron, except the AG 5 isolates, moderately sensitive to pencycuron. These results show that, although AG 3 is the most common R. solani group on potato in France, AG 5 and AG 2-1 may be present. Isolates differed for pathogenicity. In vitro sensitivity to fungicides varied among AGs.  相似文献   

4.
Two-hundred and forty-eight isolates of Rhizoctonia spp, were obtained from 13 locations in Gifu Prefecture in Japan using the plant debris particles isolation, colonization of bait tissue, and soil-clump plating methods. Of the isolates, 143 were binucleate Rhizoctonia spp., 60 were R. solani and 45 were R. zeae. Three isolates of R. solani and 54 of binucleate Rhizoctonia spp, were hypovirulent on radish, whilst all isolates of R. zeae were highly virulent, Hypovirulent strains were isolated most frequently by the plant debris particles isolation method, Hypovirulent isolates of R. solani belonged to anastomosis group 4, whilst the hypovirulent binucleate Rhizoctonia isolates belonged to AG A, AG Ba, AG G, and AG O.
Thirty-two isolates of Rhizoctotria spp, selected for hypovirulence on radish were tested on cucumber in vitro. Only five binucleate Rhizoctonia isolates and one R. solani isolate were hypovirulent on both species, and these isolates were also hypovirulent on seven other crop species. Cucumber showed wide variation in disease susceptibility to different isolates but hypovirulent isolates exhibited a consistent reaction on five different host cultivars, Pathogenicity tests using cucumber grown in soil also showed consistent reactions with isolates selected either for hypovirulence or virulence. The results support the use of cucumber in bioassays for identifying hypovirulent isolates of binucleate Rhizoctonia spp.  相似文献   

5.
Fifteen isolates of binucleate Rhizoctonia fungi (BNR) were studied as potential biocontrol agents for protection of potato from Rhizoctonia canker in artificially infested greenhouse soil and potato fields naturally infested with Rhizoctonia solani (AG-3). Eight of the BNR reduced incidence and severity of Rhizoctonia stem canker in greenhouse experiments by an average of 78 and 85%, respectively. In a field naturally infested with R. solani, selected isolates of BNR and the fungicide Tops 2.5D (thiophanate-methyl) were equally protective of potato from Rhizoctonia stem canker. BNR isolates gave protection of potato from Rhizoctonia stolon canker similar to PCNB and superior to Tops 2.5D. Cultivars Atlantic, Irish Cobbler, Kennebec, Norchip, Russet Burbank, and Superior were protected equally from Rhizoctonia stem canker by selected isolates of BNR under field conditions. Isolates of BNR show potential as biocontrol agents for protection of potato from Rhizoctonia canker.  相似文献   

6.
ABSTRACT Isolates of Rhizoctonia solani collected from mycorrhizal orchid (Pterostylis acuminata) plants and adjacent leaf litter were characterized. Of 23 selected isolates, 20 were members of a new anastomosis group (AG-12) and the rest were members of AG-6. There were no bridging anastomosis reactions observed between AG-12 and other AGs of R. solani. Among the 20 isolates of AG-12 evaluated, 18 vegetatively compatible populations were detected, indicating diversity within the AG. Mature cultures were dark brown, as were mature sclerotia. Some cultures produced alternating dark- and light-colored concentric rings, with sclerotia forming in the darker rings. Most cultures were appressed to the agar surface. In tests run to characterize pathogenic potential, selected mycorrhizal isolates of AG-12 and AG-6 did little damage to potato and barley seedlings, moderate damage to head lettuce seedlings, and more extensive damage to seedlings of cauliflower and radish. Isolates of AG-12 have not been observed to fruit in nature, and all attempts to induce formation of the teleomorph (Thanatephorus cucumeris) in the laboratory by selected isolates of AG-12 failed.  相似文献   

7.
Sixty-two isolates of Rhizoctonia spp. were collected from Belgian cauliflower fields during 2005 and 2006. The majority of the isolates (60 out of 62) had multinucleate cells and were identified as Rhizoctonia solani . Characterization of anastomosis groups (AGs) was performed using pectic zymograms, PCR-RFLP and sequencing of the rDNA-ITS region. The most prevalent AG was AG 2-1 (55% of isolates), followed by AG 2-1 subset Nt (11%), AG 1-1C (8%), AG 5 (8%), AG 4 HGII (6%), AG 3 (5%) and AG 1-1B (3%). Pathogenic potential towards different vegetable crops and towards maize was determined. Damage to cauliflower and endive was caused by different AGs, with the isolates aggressive towards cauliflower belonging to AG 2-1, AG 2-1 subset Nt, AG 4 HGII, AG 1-1C, AG 1-1B and AG 2-2, and those aggressive towards endive belonging to AG 1-1B, AG 1-1C, AG 2-1 subset Nt, AG 2-2, AG 4 HGII and AG 5. The most aggressive isolates towards bean belonged to AG 2-1 subset Nt and AG 2-2, for lettuce to AG 1-1B and AG 2-1, on carrot to AG 4 HGII and towards maize to AG 2-2. Within the isolates of AG 2-1, variability was observed in PCR-RFLP pattern and in aggressiveness towards several crops, indicating this subgroup to be heterogeneous. This is the first study concerning the occurrence of R. solani AGs causing wirestem in Belgian cauliflower fields and the first report of aggressive isolates of AG 1-1C, AG 2-1 subset Nt and AG 4 HGII associated with cauliflower.  相似文献   

8.
A new rot caused by a binucleate Rhizoctonia sp. affecting the tuberous root cortex of the domesticated yacon ( Smallanthus sonchifolius ) has been observed in Brazil. Isolates of a binucleate Rhizoctonia sp. were collected from roots with rot symptoms and characterized by the number of nuclei per cell, hyphal anastomosis, RAPD molecular markers, ITS-5·8S rDNA sequence and pathogenicity tests. All isolates had a mean of 1·9–2·2 nuclei per cell and anastomosed with the binucleate Rhizoctonia sp. AG G-tester strain. RAPD analysis was carried out between 11 isolates recovered from yacon and 11 AG (A, Ba, Bb, Bo, C, D, F, G, O, P, Q) standard testers of binucleate Rhizoctonia sp. Genetic similarities of 94·8–100% were observed among isolates of the binucleate Rhizoctonia sp. from yacon and all isolates were genetically more closely related to the AG G tester than other strains according to upgma analysis using RAPD markers. Homologies of complete ITS nucleotide sequences were 100% between binucleate isolates of Rhizoctonia sp. from yacon and the AG G tester. According to pathogenicity tests, the isolates caused typical rot symptoms of yacon tubers 90 days after inoculation  相似文献   

9.
Mazzola M 《Phytopathology》1999,89(10):920-927
ABSTRACT Changes in the composition of soil microbial communities and relative disease-suppressive ability of resident microflora in response to apple cultivation were assessed in orchard soils from a site possessing trees established for 1 to 5 years. The fungal community from roots of apple seedlings grown in noncultivated orchard soil was dominated by isolates from genera commonly considered saprophytic. Plant-pathogenic fungi in the genera Phytophthora, Pythium, and Rhizoctonia constituted an increasing proportion of the fungal community isolated from seedling roots with increasing orchard block age. Bacillus megaterium and Burkholderia cepacia dominated the bacterial communities recovered from noncultivated soil and the rhizosphere of apple seedlings grown in orchard soil, respectively. Populations of the two bacteria in their respective habitats declined dramatically with increasing orchard block age. Lesion nematode populations did not differ among soil and root samples from orchard blocks of different ages. Similar changes in microbial communities were observed in response to planting noncultivated orchard soil to five successive cycles of 'Gala' apple seedlings. Pasteurization of soil had no effect on apple growth in noncultivated soil but significantly enhanced apple growth in third-year orchard block soil. Seedlings grown in pasteurized soil from the third-year orchard block were equal in size to those grown in noncultivated soil, demonstrating that suppression of plant growth resulted from changes in the composition of the soil microbial community. Rhizoctonia solani anastomosis group 5 (AG 5) had no effect on growth of apple trees in noncultivated soil but significantly reduced the growth of apple trees in soil from third-year orchard soil. Changes in the ability of the resident soil microflora to suppress R. solani AG 5 were associated with reductions in the relative populations of Burkholderia cepacia and Pseudomonas putida in the rhizosphere of apple.  相似文献   

10.
The prevalence of Rhizoctonia spp. in European soils was determined by analysing soil samples from 282 locations. Rhizoctonia spp. were found in 68% of these samples from France, Germany, the UK, Poland, Italy, Spain, Hungary and the Czech Republic. Samples from 136 locations were further analysed by pyrosequencing. Seventy‐six percent of the isolates were Rhizoctonia solani and 24% binucleate Rhizoctonia spp. Rhizoctonia solani anastomosis group (AG) 5 was detected most frequently (25%), followed by AG 9 (16%) and AG 4 (13%). For the binucleate Rhizoctonia spp., AG E was most prevalent (13%). Rhizoctonia cerealis was not detected in soil samples. Soil type or cropping history had no effect on the type of Rhizoctonia observed. Rhizoctonia solani AG 5 was the most frequently detected AG irrespective of the previous crop. The spectrum of AGs detected was similar for France, Germany and Poland but was significantly different for the UK (= 0·0016). Finally, the baseline sensitivity towards sedaxane, a new active ingredient for seed treatment, was analysed for all isolates. The results indicate a low baseline sensitivity (average EC50 of 0·028 p.p.m.) for all Rhizoctonia AGs. No difference in sensitivity was observed with the isolates obtained from different countries.  相似文献   

11.
Rhizoctonia solani is an important pathogen of potatoes causing stem canker and black scurf. The fungus is a species complex comprised of 13 known anastomosis groups (AGs). AG3-PT is the anastomosis group frequently associated with disease in potatoes. A real-time PCR assay was designed to the rDNA ITS region of AG3-PT isolates to enable the pathogen to be detected directly in tuber and soil samples. The resulting assay was highly specific for AG3-PT, and did not amplify DNA from isolates from other AGs or subgroups of AG3. Using a bulk DNA extraction method capable of extracting from up to 250 g of soil, the assay could detect one individual sclerotium of AG3-PT (weighing 200 μg) in 250 g of soil. The AG3-PT assay was used, with assays for AG2-1, AG5 and AG8 to determine the prevalence of those AGs in UK potato soils and tubers. AG2-1 and AG3-PT were the predominant groups in tubers and soils, although AG3-PT was more frequently isolated from tubers, highlighting its importance as a potato pathogen. AG3-PT was also detected in more than half of the tuber samples tested suggesting the importance of seed borne inoculum.  相似文献   

12.
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.  相似文献   

13.
Martin FN 《Phytopathology》2000,90(4):345-353
ABSTRACT Rhizoctonia spp. were commonly recovered from the roots of strawberry plants growing in nonfumigated soil in the central coastal region of California. With the exception of one multinucleate isolate of R. solani (frequency of recovery of 0.8%), all other isolates were binucleate and were in anastomosis groups (AG) A, G, or I. AGs-A and -I were recovered from all five collection sites, whereas AG-G was recovered from only two sites. AG-A was the most commonly isolated AG, followed by AGs-I and -G. Similar levels of virulence were observed among the different AGs, but differences in virulence were observed among isolates in the same AG. Evaluating anastomosis grouping by pairing isolates recovered from strawberry with known tester isolates did not always yield a positive anastomosis reaction, even though both isolates anastomosed with other members of the same AG. Subsequent investigations with multiple isolates in the same AG from the same collection location confirmed that there was a lack of anastomosis or weak anastomosis reactions for some combinations of pairings, highlighting the need for to use multiple tester isolates or molecular techniques for AG determination. Restriction fragment length polymorphism (RFLP) analysis of a polymerase chain reaction-amplified region of the rDNA was effective for differentiating AGs. Sixteen RFLP groups were observed after cluster analysis with data for the size of the amplified products and fragment sizes after digestion with four restriction enzymes. Although each AG had isolates in multiple RFLP groups, any one individual RFLP group contained isolates of only a single AG. There was no consistent correlation between RFLP group and location of isolate collection.  相似文献   

14.
杨金红 《植物保护》2009,35(6):83-86
从新疆11种豆科作物病株上或病株根围土样中分离纯化出250个立枯丝核菌(Rhizoctonia DC),番红O KOH染色后观察细胞核数目,经测试全部菌株均为多核,用标准菌株测定融合群, 250个菌株分属为AG 1、AG 2、AG 3、AG 4和AG 5共5个融合群,出现频率分别为16.4%、33.2%、0.4%、32.4%和17.6%,营养亲合群判别结果表明,AG 1、AG 2、AG 4和AG 5下各有2个VCG,说明新疆豆科作物立枯丝核菌各主要菌丝融合群内均有不同程度的分化。  相似文献   

15.
Mazzola M  Gu YH 《Phytopathology》2002,92(12):1300-1307
ABSTRACT The induction of disease-suppressive soils in response to specific cropping sequences has been demonstrated for numerous plant-pathogen systems. The role of host genotype in elicitation of the essential transformations in soil microbial community structure that lead to disease suppression has not been fully recognized. Apple orchard soils were planted with three successive 28-day cycles of specific wheat cultivars in the greenhouse prior to infestation with Rhizoctonia solani anastomosis group (AG)-5 or AG-8. Suppressiveness to Rhizoctonia root rot of apple caused by the introduced isolate of R. solani AG-5 was induced in a wheat cultivar-specific manner. Pasteurization of soils after wheat cultivation and prior to pathogen introduction eliminated the disease suppressive potential of the soil. Wheat cultivars that induced disease suppression enhanced populations of specific fluorescent pseudomonad genotypes with antagonistic activity toward R. solani AG-5 and AG-8, but cultivars that did not elicit a disease suppressive soil did not modify the antagonistic capacity of this bacterial community. When soils were infested prior to the initial wheat planting, all cultivars were uniformly susceptible to R. solani AG-8. However, when pathogen inoculum was added after three growth-cycles, wheat root infection during the fourth growth-cycle varied in a cultivar specific manner. The same wheat cultivar-specific response in terms of transformation of the fluorescent pseudomonad community and subsequent suppression of Rhizoctonia root rot of apple was observed in three different orchard soils. These results demonstrate the importance of host genotype in modification of indigenous saprophytic microbial communities and suggest an important role for host genotype in the success of biological control.  相似文献   

16.
A buried-slide method of inducing teleomorph development was compared with a soil method for isolates of Rhizoctonia solani in anastomosis group 2. The teleomorph ( Thanatephorus cucumeris ) developed frequently in the buried-slide method. For non-self-anastomosing isolates, which cannot grow through soil, the teleomorph was observed only in the buried-slide method. One of the five soils tested was particularly favourable. The buried-slide method allows easy examination of the teleomorph with minimum disturbance to the hymenium. It has demonstrated the identity of non-self-anastomosing isolates with T. cucumeris.  相似文献   

17.
新疆北疆棉田立枯丝核菌不同菌丝融合群致病力的研究   总被引:1,自引:0,他引:1  
从新疆北疆棉区采集了典型的棉花立枯病病苗及棉田土标样686份,按常规分离方法分离得到399个分离物,从中鉴定出272个纯化的立枯丝核菌(Rhizoctonia solani Kühn)菌株。用标准菌株,通过载玻片菌丝融合试验测定,将纯化的272个菌株划归为3个菌丝融合群:即AG-2、AG-4和AG-5,分别占总菌株的6.24%、84.2%和1.1%。另有23个菌株不与任何标准菌株融合,占8.46%,说明新疆北疆棉田立枯丝核菌的优势菌系是多核丝核菌的AG-4融合群。通过从10种不同配方培养基中筛选效果好的麦芽蛋白胨(MPDA)配方培养基(Ⅱ)进行对峙培养,将纯化获得的272个丝核菌菌株,划分为6个不同的营养亲和群。将多核的不同菌丝融合群及其各营养亲和群代表菌株在3种不同主栽棉花品种上(每品种都加不接菌的对照)进行温室盆栽致病力测定,结果以AG-4对棉花的致病力最强,其次是非融合类,AG-2和AG-5虽致病,但并不致死苗,其平均病指数分别为94.9、81.4、53.1、43.5。  相似文献   

18.
Binucleate Rhizoctonia (BNR) isolates propagated for 20 days at 24°C on oat kernels and for 30 days on vermiculite amended with potato broth were recovered from an average of 62% of whole kernels, 100% of chopped kernels and 71 % of vermiculite particles within the cultures, respectively. Viability of BNR isolates 232-CG and JF-3S4-3 was higher when stored at 5 than at 24°C, and was slightly affected by the vacuum used to reduce the O2 level. After 17 weeks of storage at 5°C in air, BNR isolates 232-CG and JF-3S4-3 maintained similar viability (75% viability on whole oat kernels and 100% viability on chopped oat kernels), but in vermiculite amended with potato broth, viability of isolate 232-CG remained at 100% while that of JF-3S4-3 was 28%. In the glasshouse, BNR isolates 232-CG and JF-3S4-3 protected potato plants from Rhizoctonia canker caused by R. solani in soil maintained at 11, 17 and 23°C. Protection from Rhizoctonia canker was greater when BNR was delivered to soil than when placed on seed pieces. BNR-colonized-whole oat kernels placed in soil (15 g m of row) gave the greatest protection from Rhizoctonia canker in all experiments. In two field experiments in soil naturally infested with R. solani AG-3. the amount of BNR-colonized oat kernels was reduced from 15 g/m of row to 1-9 g m of row without affecting protection of potato plants from Rhizoctonia canker.  相似文献   

19.
ABSTRACT Isolates of Rhizoctonia spp. were obtained from rice in India during 2000-2003. Characterization by conventional techniques and polymerase chain reaction showed that from 110 isolates, 99 were R. solani and 11 were R. oryzae-sativae. Of 99 isolates identified as R. solani, 96 were AG1-IA, 1 was AG1-IB, and 2 were AG1-IC. Amplified fragment length polymorphism (AFLP) analyzes were used to determine genetic relationships in Rhizoctonia pathogen populations collected from different geographic regions. Cluster analysis based on the AFLP data separated isolates belonging to the three different intraspecific groups of R. solani AG1 and differentiated R. solani from R. oryzae-sativae. Analysis of molecular variance (AMOVA) revealed that geographic region was the dominant factor determining population structure of R. solani AG1-1A; host cultivar had no significant effect. Pathogenicity tests on Oryza sativa cv. Zenith revealed that isolates of R. solani AG1-1A and AG1-1B were more virulent than R. solani AG1-IC and R. oryzae-sativae isolates.  相似文献   

20.
由立枯丝核菌引起的马铃薯黑痣病是一种常见的土壤和种薯传播病害,在世界各地均有发生。本试验对分离纯化的67个丝核菌菌株进行融合群测定,结果表明:67个菌株都属于AG3融合群;对其中来自6个不同地域的11个菌株进行ITS测定,11个菌株的相似度为99%。8种药剂对马铃薯黑痣病菌的毒力测定结果表明,20%氟胺·嘧菌酯水分散粒剂50mg/L和25g/L咯菌腈悬浮种衣剂50mg/L的抑菌效果较好,室内抑菌效果可达100%。  相似文献   

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