JP6327416B2 - Potato scab inhibitor - Google Patents

Description

  The present invention relates to a potato scab control agent and a potato scab control method.

  The potato scab (common scab, deep scab) is infected with potato scab (Streptomyces scabiei, Streptomyces turgidiscabies, etc.) in potato (potato). This is a difficult-to-control soil disease that leads to a decline in quality by causing such problems as a decrease in sales in severe cases. The damage is widespread all over the world, and the occurrence area is 22,215ha in Hokkaido alone, which is 39% of the cultivation area (2008, pest control materials in Hokkaido). Although various measures such as crop rotation, fallow green manure cultivation, soil acidity adjustment, soil moisture adjustment, etc. have been taken as control methods, such as seed sterilization with chemical pesticides, sufficient control effects have not been obtained. In addition, although resistance varieties are bred vigorously, there is still a high demand for cultivation of conventional varieties with extremely low resistance. In warm areas, which are small-scale field crops, scab control is possible by soil disinfection using chemical pesticides. However, soil disinfection has a high control effect but is not desirable from the viewpoint of environmental burden.

  As new scab control methods using microorganisms, methods using various filamentous fungi (patent documents 1 to 4), bacteria (patent documents 5 to 10), and actinomycetes (patent document 11) have been developed so far. Has been. The potato scab fungus is greatly damaged under low soil moisture conditions, while the filamentous fungi and bacteria sometimes require high moisture for growth, and the control effect is not sufficient. In this respect, among the actinomycetes that proliferate vigorously under dry conditions like the potato scab pathogen, those having a high potato scab control activity have been desired. In addition, these microorganisms are used alone for scab control, but the application method of microorganisms alone has a problem that it is effective in sterilized soil but is not effective in natural soil. The reasons for this are as follows: (1) Unlike soil that is rich in nutrients, it is extremely poor in nutrients except in the vicinity of the rhizosphere and fresh organic matter, and exhibits the antagonism that microorganisms develop on the medium. (2) The competition with native microorganisms is intense in the vicinity of the rhizosphere and organic matter, and the added microorganisms often cannot be established.

  In addition, since the disease suppression mechanism of antagonistic microorganisms is not clear, it has been a problem that the cause of the variation in the control effect cannot be analyzed or technically improved. As one of the disease control mechanisms, it has been reported that Bacillus or Rhodococcus bacteria and Streptomyces actinomycetes produce substances having antibacterial activity against common scabs (Patent Documents 5, 11, and 12). . Surfactin, iturin A, and macrolactin A produced by Bacillus bacteria have been reported as substances having antibacterial activity against common scab (patent document 13, non-patent document 1).

  On the other hand, borrelidin, a kind of macrolide antibiotic, is a substance that suppresses Borrelia spp., A causative bacterium of tick and lice-mediated recurrent fever, from the actinomycete culture solution. Isolated. After that, this substance has angiogenesis inhibitory activity, so that it is a lead compound for anticancer agents (Non-patent Document 2) and also has an inhibitory action against Plasmodia spp. As a lead compound of malaria drugs (Non-patent Document 3), development is proceeding.

JP 2009-136216 A JP 2006-199601 A JP 2004-262801 A Japanese Patent Laid-Open No. 10-17422 JP 2007-153873 A JP 2005-289878 A Japanese Patent Laid-Open No. 2005-60287 JP 2001-327281 A Japanese Patent Publication No. 5-87044 Japanese Examined Patent Publication No. 2-48509 JP 2005-295924 A JP 2008-231023 A JP 2007-99626 A

J. et al. S. Han et al. 2005. J. et al. Appl. Microbiol. 99: 213-221. Wakabayashi et al. 1997. J. et al. Antibiotics 50: 671-676. Otoguro et al. 2003. J. et al. Antibiotics 56: 727-729.

  The subject of this invention is providing the new potato scab inhibitor and the suppression method.

  Therefore, the present inventor searched for a substance having a high antibacterial action against causative bacteria of potato scab, and found that the effect of borrelidin, a salt thereof or an ester thereof was extremely high. Moreover, it discovered that the microorganisms which produce borrelidin, its salt, or those esters also have the effect which suppresses a potato scab. Furthermore, the present inventors completed the present invention by finding that microorganisms producing borrelidins or salts thereof can be selected by culturing a sample such as soil using a medium containing borrelidins or salts thereof and selecting growing microorganisms.

That is, the present invention provides the following [1] to [5].
[1] General formula (1)

(Wherein R ¹ represents a hydroxy group or an alkoxy group)
A potato scab inhibitor containing borrelidins represented by the formula:

[2] General formula (1)

(Wherein R ¹ represents a hydroxy group or an alkoxy group)
A potato scab control method using borrelidins represented by the formula:
[3] Borreridins or salts thereof, microorganisms producing borrelidins or salts thereof, cultures of microorganisms producing borrelidins or salts thereof, or plants inoculated with microorganisms producing borrelidins or salts thereof [2] ] The potato scab control method of description.
[4] Streptomyces sp. ARDB-1 (NITE P-1162), Streptomyces sp. ARDB-2 (NITE P-1163), Streptomyces sp. ARDB-3 (NITE P-1164) or a mutant thereof.
[5] A method for selecting a microorganism that produces borrelidins or a salt thereof, comprising culturing a sample in a medium containing borrelidins or a salt thereof and selecting a growing microorganism.

  According to the present invention, the occurrence of potato scab can be remarkably suppressed by using borrelidins, salts thereof, or microorganisms that produce them.

The active ingredient of the potato scab control agent of the present invention is borrelidins represented by the above general formula (1), a salt thereof, or a microorganism that produces them. Here, borrelidins are borrelidin and its alkyl ester (R ¹ = alkoxy group).

In the general formula (1), R ¹ is a hydroxy group or an alkoxy group. Examples of the alkoxy group include a methoxy group, an ethoxy group, an n-propoxy group, an i-propoxy group, an n-butoxy group, an n-pentyloxy group, and an n-hexyloxy group, which are linear or branched alkoxy having 1 to 6 carbon atoms. Groups. Among these alkoxy groups, a linear or branched alkoxy group having 1 to 4 carbon atoms is preferable, and a methoxy group and an ethoxy group are more preferable.

In the general formula (1), the salt when R ¹ is a hydroxy group is not particularly limited, but alkali metal salts such as sodium and potassium; alkaline earth metal salts such as calcium and magnesium; ammonium salts and the like Is mentioned.

  The borrelidins (1) or salts thereof can be produced by culturing a borrelidin-producing bacterium belonging to the genus Streptomyces, and can also be produced by a known chemical synthesis method (Nagamitsu et al. 2004). Organic Letters 6: 1865-1869). Examples of borrelidin-producing bacteria include Streptomyces abikoensis, S. et al. aburaviensis, S. et al. abyssalis, S.M. achromogenes, S.M. acidiscabies, S. et al. acrimycini, S .; aculeolatus, S. et al. afghaniensis, S .; africanus, S .; alanosinicus, S.A. albaduncus, S. et al. albialialis, S. al. albidochromogenes, S. et al. albidoflavus, S. et al. albiflaviniger, S .; albieticuli, S. et al. albofaciens, S. et al. alboflavus, S. et al. albogriseolus, S. et al. albolongus, S.M. alboniger, S .; albospinus, S. et al. albosporeus, S. et al. alboilticillatus, S. et al. albovinaceus, S. et al. alboviridis, S. et al. albulus, S. et al. albus, S. et al. aldersoniae, S. et al. almquistii, S .; alni, S.A. altioticus, S. et al. amakusaensis, S .; ambfaciens, S .; aminophilus, S.M. anandii, S .; angelmyceticus, S. et al. anthocyanicus, S.H. antibioticus, S .; antimycoticus, S .; anulatus, S. et al. aomiensis, S .; arabicus, S .; ardus, S.M. arenae, S.M. argenteolus, S. et al. armeniacus, S. et al. artemisiae, S .; ascomycinus, S. et al. asiaticus, S. et al. asterosporus, S.A. atacamensis, S. et al. atratus, S. et al. attributer, S. et al. atroaurantiacus, S. et al. atroliviaceus, S. et al. atrovirens, S.M. aurantiacus, S .; aurantiogriseus, S .; auratus, S. et al. aureocirculators, S. et al. aureofaciens, S. et al. aureorectus, S. et al. aureoversilis, S. et al. aureoverticulatus, S. et al. aureus, S .; avellaneus, S .; avermetinius, S. et al. avermitilis, S. et al. aviceniae, S .; avidinii, S .; axinellae, S .; azaticus, S .; azureus, S .; baarnensis, S.M. bacilaris, S. et al. badius, S.B. baldaccii, S .; balliensis, S. et al. bambergiensis, S. et al. Bangladeshens, S .; beijianggensis, S .; bellus, S .; bikiniensis, S .; bibicularillatus, S. et al. blastmyceticus, S.M. bluesis, S. et al. bobili, S .; bottropensis, S. et al. brasiliensis, S.M. brevispora, S .; bungoensis,

Streptomyces cacaoi, S. caelestis, S. et al. caeruleatus, S. et al. caeruleus, S. et al. californicus, S .; calvus, S .; canarius, S. et al. Candidus, S. canescens, S. et al. canngkringensis, S .; caniferus, S. canus, S. et al. capilisspiralis, S. et al. capoamus, S. et al. carpaticus, S .; carpinensis, S .; castellarensis, S. et al. catenulee, S. et al. caviscabies, S .; cavourensis, S. et al. cellostaticus, S. et al. celluloflavus, S. et al. cellulolyticus, S. et al. cellulosae, S. et al. champavati, S. et al. chartreusis, S. et al. chattanoogensis, S. et al. cheonanensis, S .; chibaensis, S. et al. cholestomicus, S. et al. chromofuscus, S. et al. chryseus, S.M. chrysomalus, S. et al. cinereorectus, Streptomyces cinereolber, S. cinereospinus, S .; cinereus, S .; cinerochromogenes, S .; cinnabarinus, S .; cinnamonensis, S. et al. cinnamoneus, S .; Cirratus, S. et al. ciscacasicus, S. et al. citrefluorescens, S. et al. crafter, S.C. Clavuligerus, S .; coacervatus, S. et al. cochleatus, S .; cocklensis, S. et al. coelescens, S .; coelicoflavus, S. et al. coelicolor, S.M. coeruleoflavus, S. et al. coeruleofuscus, S. et al. coeruleoprunus, S. et al. coeuruleorubidus, S. et al. coerulescens, S. et al. collinus, S. et al. columbiensis, S. et al. corcorusii, S. et al. costaricanus, S .; cremeus, S.M. crystallinus, S. et al. curakoi, S .; cuspidosporus, S. et al. Cyaneofuscatus, St. myaneus (S. cyaneus), S. cyaneus. cyanalbus, S.C. cystargineus, S .; daghestanicus, S. et al. daliensis,

Streptomyces decanensis, S. decoyicus, S .; demaiini, S .; diastaticus, Streptomyces diastatochromogenes, S. diastatomicus. distallicus, S .; djakartensis, S.D. drozdowiczi, S .; durhamensis, S. et al. durmitorensis, S. et al. echinatus, S. et al. echinoruber, S. et al. ederensis, S. et al. ehmensis, S. et al. emiensis, S.M. endophyticus, S .; endus, S.M. enissocaesilis, S. et al. erumpens, S .; erythraeus, S .; erythrogriseus, S. et al. eurocidicus, S. et al. europaeiscabiei, S. et al. eurythermus, S. et al. exfoliatus, S. et al. fellus, S.M. Fenghuangensis, S .; ferraritis, S. et al. fervens, S.M. filamentousus, S. et al. fieldensesis, S .; filipinensis, S. et al. fibriatus, S .; fimicarius, S .; finlayi, S .; flaveolus, S. et al. flavus, S. et al. flavidofuscus, S .; flavidovirens, S .; flaviscleoticus, S. et al. flavofungini, S. et al. flavofuscus, S .; flavogriseus, S .; flavopersicus, S .; flavotricini, S. et al. flavovariabilis, S. et al. flavovirens, S .; flavoviridis, S. et al. flocculus, S. et al. floridae, S .; fluorescens, S.M. fradiae, S .; fragilis, S .; fulvissimus, S. et al. fulvorobeus, S. et al. fumanus, S .; fumigatiscleroticus, S. et al. galbus, S.M. galilaeus, S .; gancidicus, S. et al. gardneri, S .; gelaticus, S .; geldanamicininus, S. et al. geesiriensis, S .; ghanaensis, S .; gibsonii, S.M. glacescens, S. et al. glaucinger, S .; glacosporus, S. et al. glaucus, S .; globisporus, S. et al. globosus, S. et al. glomeratus, S. et al. glomeroaurantiacus,

S. glycovorans, S .; gobitricini, S .; goshikiensis, S. et al. gougerotiti, S. et al. graminearus, S .; gramineus, S .; gramanofaciens, S .; griseiniger, S .; griseinus, S .; griseaaurantiacus, S. et al. griseobrunneus, S. et al. griseocarneus, S .; grieochromogenes, S. et al. griseoflavus, S .; griseofuscus, S. et al. griseoincarnatus, S. et al. grieooloalbus, S .; griseolosporeus, S .; griseolus, S .; griseleutus, S. et al. griseemycini, S. et al. griseopranus, S. et al. grieseorubens, S.M. griseoruber, S .; griseorubibiginosus, S. et al. griseosporeus, S .; griseostramineus, S. et al. griseoverticilatus, S. et al. griseoviridis, S. et al. griseus, S .; ganduensis, S .; bulbargensis, S. et al. hachijoensis, S.H. hainanensis, S.H. haliclonae, S .; Halstedii, S.H. hawaiiensis, S. et al. hebeiensis, S.H. heilongjiensis, S.H. heliomycini, S.H. helvaticus, S.H. herbaseus, S. et al. herbariccolor, S.H. himastatinicus, S.H. Hiroshimensis, S.H. hirsutus, S. humidus, S. humiferus, S. et al. hyderabadensis, S. et al. hydrogenans, S .; hygroscopicus, S.H. hypoliticus, S.H. iakirus, S .; incanus, S.M. indiaensis, S .; indicus, S.M. indigoferus, S. et al. indonesiensis, S. et al. intermedius, S.M. inusitutus, S. et al. ipomoeae, S .; iranensis, S .; Janthinus, S .; javensis, S.J. jietaisiensis, S. et al. Kanamyceticus, S. Kashimirensis, S. Kashmirensis, S. et al. kasugaensis, S. et al. Katrae, S .; kentuckensis, S .; kifunensis,

S. kishiwadensis, S .; koyangensis, S .; kunmingensis, S .; Kursanovii, S .; labedae, S .; raceyi, S .; lacticiproductions, S .; laculatispora, S. et al. ladakanum, S .; lanatus, S. et al. lateritis, S. et al. Laurentii, S .; lavendofoliae, S. et al. lavendulae, S. et al. lavenduligrieus, S. et al. lavendulocolor, S.M. levis, S.M. libani, S .; lienmycini, S .; lilacinus, S.M. limosus, S .; lincolnensis, S .; Lipmanii, S .; litmocidin, S. et al. lomondensis, S.M. longisporovlav, S. longispoorruber, S .; longisporus, S .; longwoodensis, S .; lucense, S .; Lunalinharesii, S .; koyangensis, S .; kunmingensis, S .; Kursanovii, S .; labedae, S .; raceyi, S .; lacticiproductions, S .; laculatispora, S. et al. ladakanum, S .; lanatus, S. et al. lateritis, S. et al. Laurentii, S .; lavendofoliae, S. et al. lavendulae, S. et al. lavenduligrieus, S. et al. lavendulocolor, S.M. levis, S.M. libani, S .; lienmycini, S .; lilacinus, S.M. limosus, S .; lincolnensis, S .; Lipmanii, S .; litmocidin, S. et al. lomondensis, S.M. longisporovlav, S. longispoorruber, S .; longisporus, S .; longwoodensis, S .; lucense, S .; Lunalinharesii, S .; misakiensis, S.M. missionensis, S.M. mobaraensis, S .; monomycini, S.M. mordarskii, S .; morokaense, S .; morokaensis, S. et al. murinus, S.M. mutabilis, S. et al. mutomycini, S. et al. Naganishi, S .; nanhaiensis, S.N. nanchensis, S .; narbonensis, S.N. nashivillensis, S .; netropsis, S .; neyagawaensis,

S. niger, S.M. nigrescens, S .; nigrifaciens, S .; Nitrospores, S. niveiscabiei, S .; niveoruber, S.N. niveus, S .; noboritoensis, S .; nodosus, S .; nogalata, S.M. nojiriensis, S.M. noursei, S .; novaecaesareae, S .; ochraciscleroticus, S. et al. odorifer, S .; olivaiscleroticus, S. et al. olivaceoviridis, S. et al. olivaceus, S .; olivochromogenes, S .; olivomycini, S .; olivetriculi, S. et al. olivevertillatus, S. et al. olivoviridis, S .; omiyaensis, S. et al. orinoci, S .; osmaniensis, S.M. pactum, S. et al. panacagri, S .; paracochleatus, S. paradoxus, S .; parvisporogenes, S .; parvulus, S. et al. parvus, S .; paucusporeus, S. et al. peucetius, S .; phaeochromogenes, S .; phaefaciens, S .; phaeogriseichromatogenes, S .; phaeoleutechromatogenes, S .; phaeoluteigrieus, S. et al. phaepurpureus, S .; phaeoviridis, S. et al. pharetrae, S .; pharmamarensis, S. p. phosalacineus, S .; phytohabitans, S .; pilosus, S .; platensis, S. et al. plicatus, S .; plumbylistens, S. et al. pruricolorescens, S. et al. polybiobiotics, S. et al. polychromogenes, S. et al. poonensis, S .; praecox, S.M. pranopilosus, S. et al. prasinosporus, S. et al. prasinus, S. et al. platens, S. et al. prunicolor, S .; psamicticus, S .; pseudoechinosporeus, S. p. pseudogriseolus, S. p. pseudovenezuelae, S. p. pulveraceus, S .; puniceus, S .; puniciscabiei, S .; purpeofuscus, S. et al. purpurassens, S .; purpureus, S.P. purpurgeneiscleroticus, S. et al. qinglanensis, S .; racemochromogens, S .; radiopugnans, S .; Rameus, S .; ramulosus, S. et al. rangeonensis, S .; rapamycinus, S. et al. recifensis, S. et al. rectivellillatus, S. et al. Rectiviolaceus, S. regensis, S.M. resistomycincius, S. et al. reticuliscabiei, S. et al. rhizosphaericus, S .; rhizosphaerius, S .; rimosus,

Streptomyces resiliensis, S. rochei, S .; rosebus, S. et al. roseiscleoticus, S. et al. rosediastatics, S. et al. roseflavus, S. et al. rosefulfulus, S. et al. roseolilacinus, S. et al. roseolus, S. et al. roseosporus, S. et al. roseberticlatus, S. et al. roseioviolaceus, S. et al. roseiviris, S. et al. ruanii, S .; rubber, S.R. rubidus, S. et al. rubiginosohelvolus, S. et al. rubiginosus, S. et al. rubrogriseus, S. et al. rubrus, S.M. ruggersensis, S. et al. salmonis, S.M. sampsonii, S .; Samsunensis, S.M. sanglieri, S .; sannanensis, S.M. sanensis, S.M. sapononensis, S. et al. Scabiei, S. Scabrisporus, S. sclerotials, S. et al. scopiformis, S. et al. scopuliridis, S. et al. sedi, S. seoulensis, S. et al. septus, S. et al. seranimatus, S. et al. setae, S .; setoni, S.M. shaanxiensis, S .; shenzhenensis, S .; showdoensis, S.H. silaceus, S. et al. sindenensis, S .; sioyaensis, S. sodiphilus, S. et al. somaliensis, S.M. sparsogenes, S .; sparsus, S. et al. specialis, S. Spectabilis, S. speibonae, S .; speleomycini, S.M. spheroides, S. et al. spinoverrucosus, S. et al. spiralis, S. et al. spiroverticlatus, S. et al. spitsbergensis, S .; Spongiae, S .; sporocinereus, S.M. Sporivivatus, S. spororaveus, S. sporoverrucosus, S. et al. Staurosporinus, S. stellascabiei, S .; stramineus, S .; subtilus, S. et al. sulfofaciens, S. et al. sulphurus, S. et al. sundarbansensis, S. et al. synnematoforms, S .; syringium, S. et al. tacrolimicus, S. et al. tanashiensis, S. et al. tatyamensis,

S. tauricus, S. et al. tendae, S.M. termum, S.M. thermocalitolelans, S.M. thermoautotropicus, S. et al. thermocarboxydovorans, S. et al. thermocarboxydus, S.M. thermophilophilus, S. et al. thermodiastatics, S.H. thermogriseus, S.M. thermolineatus, S. et al. thermonitrificans, S. et al. thermospinosis porus, S. et al. thermoviolaceus, S. et al. thermovulgaris, S .; thinghirensis, S. et al. thioluteus, S. et al. torulosus, S.M. toxitricini, S. et al. tricolor, S.M. tritolerans, S. et al. tubercicus, S. et al. tuirus, S .; turgidiscabies, S. et al. umbrinus, S. et al. variabilis, S.M. variegatus, S. et al. varsoviensis, S. et al. vastus, S.M. venezuelae, S .; vietnamemensis, S. et al. Vinaceus, S.M. Vinaceus druppus, S. et al. violaceochromogens, S .; violaceolatus, S. et al. violacelectects, S. et al. violaceruber, S.M. violaceorubidus, S. et al. violaceus, S .; violaceusniger, S .; violarus, S. et al. violassens, S. et al. violatus, S.M. violens, S.M. virens, S.M. virginiae, S .; viridiflavus, S. et al. viridiviolaceus, S. et al. viridobrunneus, S. et al. viriochromogens, S. et al. viridodiastaticus, S. et al. viridoflavum, S. et al. viridosporus, S. et al. vitaminophilus, S .; wedmorens, S.W. wellingtoniae, S .; werraensis, S.W. willmoreei, S.W. xanthchromogenes, S.M. xanthocicus, S. et al. xantholiticus, S. et al. xanthophaeus, S. et al. xiamenensis, S. et al. xinghaiensis, S .; xishensis, S .; yanglinensis, S .; yanii, S .; yatensis, S .; yoochonensis, S. et al. yerevanensis, S. et al. yogyakartensis, S .; yokosukanensis, S. et al. yousoufiensis,

S. yunnanensis, S .; zamyceticus, S. et al. and microorganisms belonging to Zinciresistens. Among them, Streptomyces rochei, Streptomyces griseis, Streptomyces parvulus, Streptomyces parvulus, Streptomyces parvulus, Streptomyces calfornicus (Streptomyces calfornicus) A microorganism belonging to Streptomyces candidus is preferred.
Preferred specific examples of the microorganism used in the present invention include Streptomyces sp. ARDB-1 (NITE P-1162), ARDB-2 (NITE P-1163), ARDB-3 (NITE P-1164) strains, and mutants thereof. Here, mutants include naturally occurring mutants and mutants by ordinary physical and chemical means.

  As a method for selecting microorganisms producing the above borrelidin, microorganisms isolated from a source sample existing in any natural environment such as soil, rhizosphere soil, potato tuber surface, compost, humus soil, river water, etc. are cultured, A method for measuring the amount of borrelidin from the culture solution is mentioned. The medium used in this case is not particularly limited, but an oatmeal / casamino acid-added medium, Tryptic Soy Broth, and the like are preferable. The separation source is not particularly limited, but the rhizosphere soil of a green manure crop is preferable from the standpoint of fixing to green manure, and the potato surface and attached soil are preferable from the standpoint of fixing to potato.

  Alternatively, the sample can be inoculated into a selective medium containing borrelidin, and a microorganism capable of forming a colony can be selected as a candidate for borrelidin producing strain. In this case, the concentration of borrelidin in the medium is preferably 0.1 to 100 μg / mL, and particularly preferably 1 to 35 μg / mL.

  When these microorganisms are grown, and in order to produce borrelidins of the general formula (1) or salts thereof using these microorganisms, the culture should be carried out under conditions where normal Streptomyces microorganisms grow. . It is preferable to add a carbon source, a nitrogen source, a vitamin source, a mineral source, and the like necessary for the growth of Streptomyces microorganisms to the medium. As the carbon source, glucose, starch syrup, soluble starch and the like are used. Nitrogen sources include casamino acid, casein enzyme digest, soybean digest, oat bran, wheat bran, rice bran, yeast extract, peptone, meat extract, corn steep liquor, amino acid solution, soybean meal, ammonium sulfate, ammonium chloride, etc. Can be used. Oatmeal, whole wheat flour, etc. can be used as both a carbon source and a nitrogen source. As the mineral source, magnesium salt, potassium salt, phosphate, iron salt and the like can be used. The medium may be a solid medium or a liquid medium. In the case of liquid culture, shaking culture and stirring culture may be performed.

  The culture conditions are aerobic conditions, preferably 8 to 45 ° C. and pH 3.2 to 9.5. The culture time may be 24 to 240 hours.

As the culture grows in the medium and borrelidins or salts thereof accumulate due to the culture, these culture solutions can be used as they are. In addition, the cells and the culture solution can be separated from each other by a conventional method and used.
In addition, borrelidins or salts thereof can be partially purified or purified and isolated from a culture containing the borrelidins or salts thereof by porous synthetic adsorbent, ion exchange resin, solvent extraction, or the like.
For example, the culture solution can be adsorbed on a polystyrene synthetic adsorbent, a styrene-divinylbenzene synthetic adsorbent or a methacrylic synthetic adsorbent, and then eluted with an alcohol or ketone aqueous solution to obtain a concentrated solution.
Further, for example, the concentrated solution can be concentrated with borrelidins or salts thereof in an organic solvent by mixing and separating with an organic solvent such as ethyl acetate, diethyl ether, or n-butanol.
In the case of using the above microbial cells, it is possible to use a product obtained by recovering a precipitate by centrifugation after liquid culture and adding a commonly used protective agent to freeze-drying. Examples of the protective agent include skim milk and trehalose.

Boreridine isolates can also be converted to various alkyl esters by the following method. That is, by heating and refluxing in an alcohol (R ² OH) such as methanol, ethanol and propanol in the presence of an acid catalyst such as sulfuric acid and p-toluenesulfonic acid, using borrelidin (b-1) as a raw material. A compound (b-2) can be obtained. At this time, although there is no restriction | limiting in particular in the initial concentration of borrelidin (b-1), For example, it is preferable that it is 1-20 mass%, especially 5-10 mass%. In order to collect a product from the reaction solution after the reaction, the reaction solvent is distilled off, a product-soluble organic solvent that does not mix with water and water are added, and after recovering the organic solvent layer, an alkaline aqueous solution such as a saturated sodium carbonate aqueous solution is collected. The organic solvent is distilled off and then recrystallized from a single or mixed solvent as necessary. Moreover, what is necessary is just to isolate by HPLC as needed.

(Wherein R ² represents an alkyl group)

  Boreridines or salts thereof have an excellent effect of suppressing the growth of potato scab, and are excellent in the effect of suppressing the onset of potato scab, as shown in Examples below. Moreover, the potato scab control effect of borrelidins or salts thereof can be obtained by directly using borrelidins or salts thereof, but microorganisms producing borrelidins or salts thereof, cultures of the microorganisms, or microorganisms It can also be obtained using inoculated plants. Therefore, the potato scab control agent and the suppression method of the present invention include borrelidins or salts thereof, microorganisms that produce borrelidins or salts thereof, cultures of microorganisms that produce borrelidins or salts thereof, or borrelidins or salts thereof. Plants inoculated with microorganisms that produce salts can be used.

Borreridins or salts thereof obtained by the above, cultures containing borrelidins or salts thereof, and purified products of the cultures may be mixed with soil as they are, but powders, granules, wettable powders, emulsions Alternatively, it may be formulated using a carrier that is usually used, such as a paste.
For example, as a solid carrier, vegetable powder (wheat flour, corn grits, soybean flour, wheat bran, oat bran, starch, crystalline cellulose, rice bran, rapeseed oil cake, soybean oil cake, defatted rice bran, etc.), mineral powder (vermiculite, zeolite, Kaolin, clay, bentonite, talc, diatomaceous earth, etc.), polymer compounds (polyvinyl acetate resin, polyvinyl alcohol resin, petroleum resin, etc.), alumina, waxes and the like can be used. As the liquid carrier, for example, alcohols (methanol, ethanol, propanol etc.), ethers (dioxane, tetrahydrofuran etc.), esters (ethyl acetate, butyl acetate etc.), water or the like can be used.

  When the potato scab control agent of the present invention is used as an aqueous solution or suspension, the pH is preferably 2 to 8. Buffers for adjusting the pH include acetic acid, citric acid, fumaric acid, apple Examples thereof include organic acid salts such as acid, lactic acid, gluconic acid and tartaric acid, inorganic salts such as phosphoric acid, hydrochloric acid and sulfuric acid, hydroxides such as sodium hydroxide and potassium hydroxide, ammonia or aqueous ammonia, etc. Or you may use in combination of 2 or more types, and also may combine with another pH adjuster suitably.

  The potato scab control agent of the present invention may be subjected to soil treatment as it is, or may be mixed with agricultural chemicals, fertilizers, soil improvement materials, compost, and the like. In particular, as a pesticide, DD agent having potato scab control effect, oxytetracycline agent, streptomycin agent, oxolinic acid agent, kasugamycin agent, carbam agent, thiofamate methyl agent, dazomet agent, tolcrophos methyl agent, fursulfamide agent, Needless to say, it is desirable to use fluazinam and copper wettable powder in combination.

In addition, the concentration of borrelidins or salts thereof obtained by the above, the culture containing the borrelidins or salts thereof, or the purified product of the culture used for mixing with soil is the concentration of borrelidins in soil or The concentration of the salt can be in the range of 0.01 to 100 ppm, more preferably 0.1 to 10 ppm. In this case, it may be mixed in advance before sowing, or may be sprayed or irrigated during the growth period.
There is no particular restriction on the administration time, but it is particularly effective to treat it immediately before potato planting or in the tuber formation period.

Microorganisms that produce borrelidins or salts thereof may be mixed directly with soil, inoculated into potatoes (seed potatoes), or inoculated into plants.
When applied directly to the soil, the cells may be mixed with the soil as it is or mixed with the culture solution, but commonly used carriers such as powders, granules and wettable powders may be added. You may formulate using.
For example, as a solid carrier, vegetable powder (wheat flour, corn grits, soybean flour, wheat bran, oat bran, starch, crystalline cellulose, rice bran, rapeseed oil cake, soybean oil cake, defatted rice bran, etc.), mineral powder (vermiculite, zeolite, Kaolin, clay, bentonite, talc, diatomaceous earth, etc.), polymer compounds (polyvinyl acetate resin, polyvinyl alcohol resin, petroleum resin, etc.), alumina, waxes and the like can be used. Among these, oat bran, wheat bran and rice bran are preferable. Moreover, as a liquid carrier, water etc. can be used, for example.
The pH when the microorganism is an aqueous solution or suspension is preferably 5 to 8, and the buffer for adjusting the pH is acetic acid, citric acid, fumaric acid, malic acid, lactic acid, gluconic acid, Examples include organic acid salts such as tartaric acid, inorganic salts such as phosphoric acid, hydrochloric acid and sulfuric acid, hydroxides such as sodium hydroxide and potassium hydroxide, ammonia or aqueous ammonia, and these are used alone or in combination of two or more. It may be combined with other pH adjusters as appropriate.
When the above microorganism is used for mixing with soil, the number of microorganisms in the soil is 10 ⁵ to 10 ⁹ c.g per dry soil. f. u. (Colony forming unit, hereinafter CFU), more preferably in the range of 10 ⁷ to 10 ⁸ CFU. In this case, it may be mixed in advance before sowing, or may be sprayed or irrigated during the growth period.
There is no particular restriction on the administration time, but it is particularly effective to treat it immediately before potato planting or in the tuber formation period.

Moreover, when inoculating said microorganisms into a potato, the number of the said microorganisms in a solution can be 10 < ⁶ > -10 < ⁸ > CFU per mL, More preferably, it can be 10 < ⁸ > CFU. In this case, a spreading agent, such as carboxymethylcellulose, can be added to the solution and seed potatoes can be immersed and planted.

It is also possible to inoculate a plant with the above-mentioned microorganism and use the plant by mixing it. In this case, the seed of the plant may be inoculated, the plant may be inoculated while growing, or the plant may be grown and then inoculated after cutting the ground part. Soil mixing may be performed while the plant is growing, or may be performed after cutting down. In that case, it is desirable to chop the above-ground part of the plant with a chopper, mower or the like before mixing with the soil. When inoculating during or after plant growth, the above microorganisms suspended in water can be sprayed with a sprayer for spraying agricultural chemicals that is usually used. In this case, the microorganism density 1mL per 10 ⁷ to 10 ¹⁰ CFU in a spray solution, more preferably in the range of ^{10 8} ~10 10 CFU. Moreover, the amount of spray liquid can be in the range of 100 to 10,000 L, more preferably 400 to 1,000 L per 1 ha of land area.
Although the plant used in this case is not particularly limited, for example, those generally used as green manure crops are preferable. Specific examples include wild oat species (Avena trigosa), oats (Avena sativa), crimson clover (Trifolium incarnatum), white pepper (Sinapis alba), hybrid sorghum (Sorghum bicolor ss ce ss). Red clover (Trifolium platense), Hazelaria (Phacelia tanacetifolia), Woolly podbetch (Vicia villosa ssp. Daiscarpa), Sudangrass (Sorghum sudanense), Himawari Zea mays, Italian ryegrass (Lolium multiflorum), centipede grass (Eremochloa ophyluroides), Naginata gay (Vulpia myuros), hairy vetch (Vicia villosa), Astragalus, Astragaus. , S. cannabina, Dicandra milantha, Guinea grass (Panicum maximum), Annual ryegrass (Lolium multiflorum), Bahiagrass (Cyonodon dactylon), Tall fescue (Fes) uca arundinacea), Kentucky bluegrass (Poa platensis), white clover (Trifolium repens), triticale (× Triticoscale), buckwheat (Fagopyrum esculentum), marigold (Tagetes emu), (Echinochlo esculenta), Ebusa (Senna obtusifolia) and the like. In addition, sweet corn (Zea mays) is preferable in the same manner as a green manure crop because the above-ground part can be mixed with soil after harvesting ears. Among these, wild oats (Avena trigosa), oats (Avena sativa), hybrid sorghum (Sorghum bicolor × S. Sudanense), Sudangrass (Sorghum sugiense), sunflower (Helianthus lime) (Zea mays), Italian ryegrass (Lolium multiflorum), centipede grass (Eremochloa ophiuroides), Naginata gay (Vulpia myuros), Guinea grass (Panicum maxumum), Annual ryumrum (ulolium mulberry) lon), tall fescue (Festuca arundinacea), Kentucky bluegrass (Poa platensis), triticale (× Triticoscale), barley (Hordeum vulgare), wheat (Triticum aestivum), and particularly preferred (Echinoculo).

  EXAMPLES Next, although an Example is shown and this invention is demonstrated further in detail, this invention is not limited to a following example.

Production Example 1 (Production of borrelidin)
A medium containing 20 g / L of oatmeal (manufactured by The Quaker Oats Company) and 1 g / L of casamino acid was adjusted to pH 7.2, and then autoclaved, and 2.5 L each was dispensed into three 3 L jars. Streptomyces sp. Precultured in this medium in advance with Tryptic Soy Broth (Becton Dickinson & Company, TSB). ARDB-1 strain was inoculated. The culture temperature was 27 ° C., and aeration culture was performed with a jar fermenter (TS-M3L type mini jar fermenter system, manufactured by Takasugi Seisakusho Co., Ltd.) at a rotation speed of 100 rpm for 10 days. The obtained culture broth was roughly purified as follows. The culture solution was centrifuged at 6,500 rpm for 30 minutes, and the supernatant was adjusted to pH 3.0 with hydrochloric acid. Separately, styrene-divinylbenzene synthetic adsorption resin Diaion HP20 was washed with propanol, then packed in a column (inner diameter 450 mm × length 550 mm), and then adjusted by passing pH 3.0 acetic acid solution through. Borrelidin was adsorbed by flowing the supernatant liquid through the column. The column was washed with 2 L of 30% by volume isopropanol, and then borrelidin was eluted with 3 L of 99% by volume isopropanol. The eluate was concentrated to about 500 mL with an evaporator, and borrelidin was extracted by repeating mixing and stirring with ethyl acetate three times. The ethyl acetate layer was dehydrated with anhydrous magnesium sulfate, silica gel (Wakogel C100, manufactured by Wako Pure Chemical Industries) was added, and then the solvent was distilled off. This silica gel was layered on top of a silica gel column (inner diameter 20 mm × length 250 mm) separately suspended and packed in n-hexane. This column was washed with 1 L of n-hexane: ethyl acetate = 6: 4 mixture, and then borrelidin was eluted with 1 L of n-hexane: ethyl acetate = 2: 8 mixture. The eluate was concentrated and dried, dissolved in a small amount of 10% by volume methanol, separately washed with methanol, and then passed through a SepPak tC18 cartridge adjusted by passing 10% by volume methanol to adsorb borrelidin. The cartridge was washed with 20 mL of 40 volume% methanol, and then borrelidin was eluted with 20 mL of 99 volume% methanol.

This sample was purified using HPLC [column, YMC ODS-A inner diameter: 10 mm × length: 250 mm (manufactured by YMC); mobile phase, 80% by volume methanol; flow rate, 3.0 mL / min], and fraction corresponding to borrelidin retention time. Minutes (8-14 minutes) were collected. Further, the fraction collected was purified by HPLC (column, YMC ODS-A inner diameter 10 mm × length 250 mm; mobile phase, 60 vol% acetonitrile; flow rate, 3.0 mL / min), and fraction corresponding to borrelidin retention time. (13 to 16 minutes) was collected. Further, the fraction collected was purified using HPLC (column, YMC Phe inner diameter 10 mm × length 250 mm; mobile phase, 60 vol% methanol; flow rate, 3.0 mL / min), and a single fraction corresponding to the borrelidin retention time. Fractions showing peaks (9-18 minutes) were collected. The ultraviolet absorption maximum of this peak was 259 nm, which was consistent with borrelidin (manufactured by Alexis, Hokkaido Wako Pure Chemicals) sold as a reagent. The obtained fraction was concentrated to dryness and dried under reduced pressure in a desiccator in the presence of diphosphorus pentoxide. As a result, 3.1 mg of borrelidin crystals were obtained. When this crystal was subjected to mass spectrometry by EMS-MS (negative ion), a deprotonated molecule (MH)-was observed at m / z 488.3002. As a result of calculating the composition of this mass number, C ₂₈ H ₄₃ NO ₆ was obtained with an error of 1.6 mDa, which was consistent with borrelidin. The chemical shifts of ¹ H-NMR (600 MHz) and ¹³ C-NMR (150 MHz) are shown in Table 1, and this content supported the chemical structure of borrelidin. From this result, the concentration of borrelidin in the culture broth was calculated to be 0.4 mg / L.

  From these results, Streptomyces sp. The ARDB-1 strain was confirmed to have the ability to produce borrelidin, and it was confirmed that borrelidin can be produced by producing a borrelidin-containing culture by culturing this strain and further purifying it.

Production Example 2 (separate strain)
300 mL of the medium shown in Preparation Example 1 was placed in a 500 mL baffle Erlenmeyer flask, sterilized by autoclave, and then Streptomyces sp. The ARDB-2 strain was inoculated and cultured with shaking at a rotation speed of 60 rpm for 10 days. The culture solution was centrifuged as in Production Example 1 and roughly purified as follows. The supernatant was adsorbed by passing through a HP20 column (inner diameter 20 mm × length 250 mm) prepared in the same manner as in Production Example 1, and then washed with 500 mL of 30% by volume isopropanol, and then washed with 500 mL of 99% by volume isopropanol. Was eluted. The eluate was concentrated with an evaporator, and borrelidin was extracted by repeating mixing and stirring with ethyl acetate three times. The ethyl acetate layer was dehydrated with anhydrous magnesium sulfate, concentrated, and purified by SepPak tC18 in the same manner as in Production Example 1. This crude product was analyzed using HPLC [column, YMC ODS-A inner diameter 10 mm × length 250 mm (manufactured by YMC); mobile phase, 80 vol% methanol; flow rate, 3.0 mL / min]. A peak was observed at a retention time of 10.5 minutes, which almost coincided with the retention time of borrelidin. Further, the ultraviolet absorption maximum was 258 nm, which was almost the same as that of borrelidin. From the peak area, the concentration of borrelidin in the medium was calculated to be 0.2 mg / L.
From the above results, it was confirmed that the ARDB-2 strain has the ability to produce borrelidin.

Production Example 3 (Type of medium)
A 500 mL baffle Erlenmeyer flask with TSB, a mixture of 2% oatmeal (The Quaker Oats Company) and 0.1% casamino acid, 2% oatmeal and soyton (Soytone, Becton Dickinson & Company) 0. 300 mL of each medium mixed with 3% was prepared, adjusted to pH 7.2, and then autoclaved. Here, Streptomyces sp. ARDB-1 strain was inoculated. The culture temperature was 27 ° C., and the mixture was cultured with shaking at 80 rpm for 7 days to obtain a mixture of borrelidin and borrelidin-producing bacteria. This was further centrifuged at 6,500 rpm for 30 minutes to obtain a borrelidin-containing liquid.
In order to measure the concentration of borrelidin in these borrelidin-containing solutions, the supernatant of the culture solution was roughly purified in the same manner as in Production Example 2, and HPLC [column, Jupiter C18 300Å inner diameter 10 mm × length 250 mm (manufactured by phenomenex); Phase, 1% by volume acetic acid-containing 70% by volume methanol; flow rate, 3.0 mL / min], quantified by the area of the peak corresponding to borrelidin (retention time, 11.2 minutes; UV absorption maximum, 259 nm). went. The results are shown in Table 2.

  From this result, it became clear that a culture solution containing borrelidin-producing bacteria and borrelidin can be prepared using various media as described above.

Production Example 4 (Oatmeal concentration)
A 500 mL baffle Erlenmeyer flask with oatmeal (The Quaker Oats Company) 0.5%, 1.0%, 1.5%, 2.0%, 2.5%, 3.0% and casamino acid 0. 300 mL of each medium mixed with 1% was prepared, adjusted to pH 7.2, and then autoclaved. Here, Streptomyces sp. ARDB-1 strain was inoculated. The culture temperature was 27 ° C., and the mixture was cultured with shaking at 80 rpm for 7 days to obtain a mixture of borrelidin and borrelidin-producing bacteria. This was further centrifuged at 6,500 rpm for 30 minutes to obtain a borrelidin-containing liquid.
In order to measure the concentration of borrelidin in these borrelidin-containing solutions, the supernatant of the culture solution was subjected to crude purification and HPLC quantification in the same manner as in Production Example 3. The results are shown in Table 3.

  From this result, it became clear that the oatmeal concentration is preferably 1 to 2% in order to make borrelidin highly contained.

Production Example 5 (Bacteria skim milk FD)
The culture solution prepared in the same manner as in Production Example 1 was centrifuged, suspended in 10% by weight skim milk in the precipitated cells, and then lyophilized. After storing at 4 ° C., the number of bacteria in the lyophilized product was measured by a dilution plate method. Specifically, 100 μL of each lyophilized product suspended in sterilized water and 100 μL of each diluted solution was added to a starch medium (15 g agar, 10 g starch, 1 g yeast extract, 1 g sucrose, 0.1 g potassium chloride, 0.1 g hydrogen phosphate per liter medium) Potassium 0.1 g, magnesium sulfate heptahydrate 0.1 g, and potassium nitrate 0.1 g were dissolved and autoclaved and applied. Table 4 shows the results of counting the number of colonies after culturing at 25 ° C. for 7 days.
The number of bacteria in the freeze-dried product was 10 ⁵ CFU / g, and it was confirmed that freeze-dried cells can be produced by this method.

Production Example 6 (HP20 crude product)
A medium containing 10 g / L oatmeal (manufactured by The Quaker Oats Company) and 1 g / L casamino acid was adjusted to pH 7.2, then autoclaved, and 2.5 L each was dispensed into three 3 L jars (total 7 .5L). Streptomyces sp. Precultured in this medium in advance with TSB. ARDB-1 strain was inoculated. The culture temperature was 27 ° C., and aeration culture was performed with a jar fermenter (TS-M3L type mini jar fermenter system, manufactured by Takasugi Seisakusho Co., Ltd.) at a rotation speed of 100 rpm for 10 days. The obtained culture solution was centrifuged at 6,500 rpm for 30 minutes, and the supernatant was adjusted to pH 3.0 with hydrochloric acid. Separately, styrene-divinylbenzene synthetic adsorption resin Diaion HP20 was washed with propanol, then packed in a column (inner diameter 450 mm × length 550 mm), and then adjusted by passing pH 3.0 acetic acid solution through. Borrelidin was adsorbed by flowing the supernatant liquid through the column. The column was washed with 2 L of 30% by volume isopropanol, and then borrelidin was eluted with 3 L of 99% by volume isopropanol. The eluate was concentrated to about 500 mL with an evaporator.
As a result of crude purification and HPLC quantification of the borrelidin concentration in this concentrated liquid in the same manner as in Production Example 3, it was found to be 16.3 mg / L.

Production Example 7 (Vermiculite mixed inoculation source)
Autoclave back (300 mm x 610 mm) with 2 L vermiculite oatmeal medium (mixed with 5.14 g oatmeal ground per 1 L vermiculite and 350 mL 0.073 mass% casamino acid) with Milliceal [Azuwan Co., Ltd.] ) And autoclaved (121 ° C., 60 minutes for two days or more). To this medium, Streptomyces sp. ARDB-1 strain was inoculated with agar pieces and cultured at 25 ° C. for 6-8 weeks. 10 g of the culture was suspended in 90 mL of sterilized water and shaken at room temperature for 20 minutes, followed by a dilution plate method.
The number of bacteria in the culture is 3.2 × 10 ^{8 to} 2.3 × 10 ⁹ CFU / g dry soil, and even when this culture is stored at 4 ° C. for 5 weeks, the number of viable bacteria is similar. confirmed. From this, it was confirmed that this method can produce a vermiculite mixed inoculation source containing bacterial cells.

Example 1 (proof of the effect of borrelidin)
Borrelidin obtained in Production Example 1 and Borrelidin sold as a reagent (Alexis, Hokkaido Wako Pure Chemicals) 1 mg each were diluted in 1 mL of ethanol, 10 μL and 100 μL were added to a filter paper disk having a diameter of 8 mm, and a vacuum desiccator. (The amount of borrelidin per disc is 10 μg and 100 μg, respectively). These discs were placed on a starch medium inoculated with the potato scab fungus Streptomyces turgidiscabies 94-3 and cultured at 27 ° C. for 3 days. The size of the inhibition circle after culture (3 repetitions) is shown in Table 5.
As a result, both the commercially available borrelidin and the borrelidin obtained in Production Example 1 formed a clear inhibition circle, and thus it was clarified that they have an inhibitory effect on the growth of potato scab.

Example 2 (Proof of effect in pot test of two strains)
A potato scab (S. turgidiscabies strain 94-3) was mixed with a medium (black soil 2.4 L, vermiculite 1.2 L, fusuma 1.2 L, oatmeal 60 g, and distilled water 1 L, and 14 to 16 g of glass lime was added. PH was adjusted to 7.2, and autoclaved at 121 ° C. for 60 minutes and cultured twice for 1 day or more), and field soil (black box soil, river sand, peat moss in a volume ratio of 7. 7: 2.3: 5) to prepare scab artificially contaminated soil (the number of scabs is 7.4 × 10 ⁶ CFU / g dry soil). Streptomyces sp. ARDB-1 and ARDB-2 strains were cultured in vermiculite oatmeal medium and mixed with 5 or 10% by volume of scab artificially contaminated soil. These soils were packed in a 3L non-woven pot [(money) Momoki Shoten], and the potato variety "Baron potato" was planted, and the entire pot was buried in a field (Hokkaido Memuro-cho) and cultivated. Table 6 shows the results of investigating the occurrence of scab in new tubers after 3 months.
In the pots applied with ARDB-1 and ARDB-2 strains, the occurrence of potato scab was reduced. Therefore, by mixing the vermiculite oatmeal medium culture of microorganisms having the ability to produce borrelidin with soil, It became clear that the onset of rickets could be suppressed.

Example 3 (Proof of effect in field test)
Streptomyces sp. ARDB-1 strain vermiculite oatmeal medium culture (the number of bacteria is about 1.2 × 10 ⁹ CFU / g dry soil) in the field of scab disease contamination (Hokkaido Memuro Town, the pathogen is S. turgidiscabies) potato rhizosphere 10% by volume was mixed in the area (30 cm × 30 cm × 15 cm per strain), and a potato variety “Baron potato” was planted. Table 7 shows the results of investigating the occurrence of scab in newborn tubers after 3 months.
In the ARDB-1 strain application zone, the occurrence of potato scab was reduced, so that the potato scab could be reduced even in the field by mixing vermiculite oatmeal medium culture of microorganisms capable of producing borrelidin with soil. It became clear that the disease can be suppressed.

Example 4 [Proof of effect of bacterial cells only (no borrelidin)]
500 mL of TSB medium was placed in each of 8 Erlenmeyer flasks with 2 L baffles, sterilized by autoclave, and then Streptomyces sp. The ARDB-1 strain was inoculated and cultured with shaking at 25 ° C. and a stirring speed of 120 rpm / min for 3 days. The culture was centrifuged at 8,000 rpm for 10 minutes, the supernatant was discarded, and the cells were washed by repeatedly suspending the precipitate in sterilized water. The cells were suspended in 400 mL of sterilized water and crushed with a homogenizer [Ace homogenizer AM-3, manufactured by Nippon Seiki Seisakusho]. After centrifuging 200 mL of the cell disruption solution, the precipitate was suspended in 150 mL of 0.1% by mass carboxymethylcellulose (hereinafter referred to as CMC), and the seed potato (variety “Baron Imo”) cut in half was immersed therein. Further, the remaining 200 mL of the microbial cell disruption solution was centrifuged, the precipitate was suspended in 540 mL of 0.1% CMC, and 105 mL each was mixed with 300 mL of vermiculite. Planted seed potato dipped in a 3L non-woven pot with scab artificially contaminated soil prepared in the same manner as in Example 2 (the number of scab pathogen is 2.0 × 10 ⁴ CFU / g dry soil). It was. Also, 10% by volume of vermiculite mixed with the bacterial cell disruption solution was mixed with the scab artificially contaminated soil, packed in a pot, and planted with potatoes (variety “Baron Imo”). As controls, a 0.1% CMC soaked potato seeding group and a 0.1% CMC-added vermiculite application group were provided. Table 8 shows the results of investigating the degree of occurrence of scab in the new tuber after 3 months after burying each pot in a field (Hokkaido Memuro-cho) and cultivating potato.
In a pot in which ARDB-1 strain liquid culture cells were mixed with soil or treated with seeds, the occurrence of potato scab was reduced. By applying microbial cells having the ability to produce borrelidin, It became clear that the onset of the disease can be suppressed.

Example 5 (proof of effect by combined use with green manure)
Potato scab (S. turgidiscabies 94-3 strain) was cultured in vermiculite oatmeal medium and mixed with field soil to produce a scab artificially contaminated soil (scattle bacterium was 4.6 × 10 ⁶ CFU / g dry soil). Streptomyces sp. The ARDB-1 strain was also cultured in the same manner and mixed with 10% by volume of the scab artificially contaminated soil (the number of the bacteria was 1.5 × 10 ⁷ CFU / g dry soil). These soils were packed in 3 L non-woven pots, seeded with wild oats (Avena trigosa) and oats (Avena sativa) and cultivated in a greenhouse (18 ° C., 14 hours light period / 10 hours dark period) for 2 months. The oat wild species, oat roots and leaves were shredded and mixed with the soil, and then left for 3 weeks while supplying water. Oat wild species and oats were sown again, and the potato variety “Baron potato” was planted in soil that had been repeatedly cultivated and mixed with soil, and the occurrence of scab disease in the new tuber was investigated three months later. The results are shown in Table 9.
Wild oat species and oat cultivation are known to have the effect of reducing the common scab of potatoes, but it is possible to obtain a higher disease control effect by combining borrelidin-producing microorganisms than these green manures alone. Became clear.

Example 6 (effect by combined use with crops)
Streptomyces sp. ARDB-1 strain was cultured in vermiculite oatmeal medium, mixed with field soil and packed in a 3 L non-woven pot (the number of bacteria was 1.4 × 10 ⁷ CFU / g dry soil), wild oat (Avena trigosa), Wheat (Triticum aestivum), potato (Solanum tuberosum), sugar beet (Beta vulgaris) and soybean (Glycine max) were cultivated and cultivated in a greenhouse (18 ° C., light period 14 hours / dark period 10 hours) for 4 months. DNA was extracted from the collected rhizosphere soil, and a competitive Qprobe-PCR method using the ITS region of the ribosomal RNA gene of the fungus as a primer was performed to quantify the copy number of the fungal gene. Table 10 shows the number of the bacterial genes increased or decreased during the cultivation period of various crops.
In the rhizosphere soil of wild oat (Avena trigosa), wheat, and potato, the number of genes of the ARDB-1 strain was increased, so the combined use of these crops increased the number of inoculated borrelidin-producing microorganisms, It became clear that there was an effect which improves the fixity to soil.

Production Example 8 (Synthesis of borrelidin methyl ester)
10 mL of methanol was added to 500 μg of borrelidin (manufactured by Alexis, Hokkaido Wako Pure Chemicals), 20 μg of paratoluenesulfonic acid was added while stirring at room temperature, and the mixture was heated to 62-64 ° C. and stirred for 2 hours. After cooling, the pH was adjusted to 8.0, and water and ethyl acetate were added for liquid separation. The ethyl acetate phase was dehydrated with anhydrous magnesium sulfate and dried, and then the ethyl acetate was distilled off under reduced pressure. This fraction was subjected to HPLC under the same conditions as in Production Example 3, and unreacted borrelidin (eluted with a retention time of 10 to 12.5 minutes) and synthesized borrelidin methyl ester (retention time of 13 to 15.5 minutes). Elution, peak top retention time 14.1 min; ultraviolet absorption maximum 261 nm). The borrelidin methyl ester fraction was dried under reduced pressure, and as a result, the yield was 70 μg.

Example 7 (proof of the effect of borrelidin ethyl ester)
50 μg of borrelidin methyl ester obtained in Production Example 8 was dissolved in 500 μL of ethanol, 100 μL was added to a filter paper disk having a diameter of 8 mm, and dried in a vacuum desiccator (the amount of borrelidin methyl ester per disk was 10 μg). . In the same manner as in Example 1, the disc was examined for inhibitory circle-forming activity against potato scab on a starch medium. The results are shown in Table 11.
Since borrelidin methyl ester also forms a clear inhibition circle, it has been clarified that it has an inhibitory effect on the growth of potato scab.

Example 8 (Selection method of borrelidin producing bacteria from soil)
Oats were planted twice, and then the soil was collected from the field (Tsujo City, Miyazaki Prefecture) where the sorghum variety "Tuchitaro" [Snow Brand Seed Co., Ltd.] was cultivated, and the plant residue was removed with a 2 mm x 2 mm sieve . After suspending 10 g of soil in 90 mL of sterilized water and shaking at 25 ° C. for 30 minutes, a serial dilution was prepared. On the other hand, diluted PTYGA medium (0.25 g of bactoprotease peptone (manufactured by Difco) per liter of medium, 0.25 g of bactotryptone (manufactured by Difco), 0.5 g of yeast extract (manufactured by Difco), 0.5 g of glucose, 30 mg of magnesium sulfate heptahydrate and 3.5 mg of calcium chloride dihydrate were dissolved and adjusted to pH 7.0, then 15 g of agar was added and sterilized by autoclave]. Borrelidin dissolved in ethanol (5 mg / mL) ) Was added in an amount of 100 μL per plate, and quickly applied uniformly using a congeal rod, and the solution was air-dried. The medium was inoculated with the above-mentioned soil suspension, and after culturing at 30 ° C. for 8 days, colonies of borrelidin-resistant actinomycetes that had grown were isolated. The isolated colonies were separately transplanted into a diluted PTYGA medium and potato scab B. The suspension of the turgidiscabies Sy9113 strain was spray-inoculated, and the presence or absence of antibacterial activity was confirmed by forming a growth inhibition circle. About 28 strains which showed antibacterial property, it culture | cultivated by the culture medium which made the oatmeal 1.0% in manufacture example 4, the supernatant of a culture solution was roughly refine | purified similarly to manufacture example 3, and quantified by HPLC. As a result, it was confirmed that one strain produced 2.85 μg / L of borrelidin.
From the above, it was revealed that borrelidin-producing bacteria can be efficiently selected by using a borrelidin-added medium.

Example 9 (Proof of effect by crudely purified culture broth)
The soil with severe potato scab disease in the Obihirogawa west district was collected, air-dried, coarse particles were removed with a 5 mm × 5 mm sieve, and filled into a 1 / 2000a Wagner pot. On the other hand, a potato variety “Baron potato” that had been cut into two pieces and bathed was cultivated and cultivated in a greenhouse. Seven days after emergence, the same soil was added at a height of 10 cm. A two-fold dilution of the crude culture solution prepared as in Production Example 6 at a rate of once every two weeks after the start of cultivation was irrigated with 500 mL per pot, and 500 mL of water was irrigated in the control group (each 2 iterations). This treatment was performed 4 times in total. In order to induce the development of potato scab, the method of irrigation during cultivation was repeated after confirming that the lower leaves were slightly deflated. Table 12 shows the results of sampling tubers after the above-ground part withered and counting the lesions that were depressed due to potato scab.
It was clarified that potato scab can be suppressed by irrigation during growth even with only HP20 crude purified product containing borrelidin.

Example 10 (Proof of effect by fungus body-coated green manure seed)
Streptomyces sp. Precultured with TSB in the medium of oatmeal 1.0% in Production Example 4 in advance. The ARDB-1 strain was inoculated and cultured with shaking at 27 ° C. and a rotation speed of 80 rpm for 7 days. The culture solution was centrifuged, and the precipitated cells were suspended in a small amount of sterile water. The seeds of wild oat (Avena trigosa) were immersed in the cell suspension for 10 minutes and then dried in the shade. A scab artificially contaminated soil prepared in the same manner as in Example 5 (scab pathogen is S. turgidiscabies KHH1-1-3 strain, 2.0 × 10 ⁴ CFU / g dry soil) is packed in a 3 L non-woven pot, Oat seeds soaked in the suspension were sown. As a control, a seed group of wild oat seeds not immersed in the cell suspension and a group in which the cell suspension was dripped onto the soil were provided. Each pot is buried in a field (Miyagi Prefecture Miyakonojo City). After 2 months, the roots and leaves of wild oat species are shredded and mixed with the soil. After standing for 3 weeks, the potato variety "Nishiyutaka" is planted in all pots. It was. Table 13 shows the results of investigating the occurrence of scab in newborn tubers after 3 months.
Although the amount of bacterial cells adhering to the seeds is very small, it has been clarified that the use of wild oat seeds immersed in the bacterial cell suspension has a higher disease control effect than the use of wild oat seeds. It was.

Claims (5)

General formula (1)

(Wherein R ¹ represents a hydroxy group or an alkoxy group)
A potato scab inhibitor containing borrelidins represented by the formula: General formula (1)

(Wherein R ¹ represents a hydroxy group or an alkoxy group)
A potato scab control method using borrelidins represented by the formula:   3. A borrelidin or a salt thereof, a microorganism producing a borrelidin or a salt thereof, a culture of a microorganism producing a borrelidin or a salt thereof, or a plant inoculated with a microorganism producing a borrelidin or a salt thereof. Potato scab control method. A microorganism that produces borrelidins or salts thereof is Streptomyces sp. ARDB-1 (NITE P-1162), Streptomyces sp. ARDB-2 (NITE P-1163), Streptomyces sp. The potato scab inhibitor according to claim 1, which is ARDB-3 (NITE P-1164) or a mutant thereof. A microorganism that produces borrelidins or salts thereof is Streptomyces sp. ARDB-1 (NITE P-1162), Streptomyces sp. ARDB-2 (NITE P-1163), Streptomyces sp. The potato scab control method according to claim 2 or 3, which is ARDB-3 (NITE P-1164) or a mutant thereof .