TR2021013538A2 - A MICROBIAL FERTILIZER AND BIOPESTICIDE FORMULATION WITH SOLID CARRIER - Google Patents

A MICROBIAL FERTILIZER AND BIOPESTICIDE FORMULATION WITH SOLID CARRIER

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TR2021013538A2
TR2021013538A2 TR2021/013538A TR2021013538A TR2021013538A2 TR 2021013538 A2 TR2021013538 A2 TR 2021013538A2 TR 2021/013538 A TR2021/013538 A TR 2021/013538A TR 2021013538 A TR2021013538 A TR 2021013538A TR 2021013538 A2 TR2021013538 A2 TR 2021013538A2
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solid carrier
formulation
microbial fertilizer
feature
bacteria
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TR2021/013538A
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Turkish (tr)
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Şenol Kotan Merve
Kotan Recep
Di̇kbaş Nesli̇han
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Atatuerk Ueniversitesi Rektoerluegue Bilimsel Arastirma Projeleri Bap Koordinasyon Birimi
Atatürk Üni̇versi̇tesi̇ Rektörlüğü Bi̇li̇msel Araştirma Projeleri̇ ( Bap ) Koordi̇nasyon Bi̇ri̇mi̇
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Priority to TR2021/013538A priority Critical patent/TR2021013538A2/en
Publication of TR2021013538A2 publication Critical patent/TR2021013538A2/en
Priority to PCT/TR2022/050916 priority patent/WO2024058727A1/en

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    • CCHEMISTRY; METALLURGY
    • C05FERTILISERS; MANUFACTURE THEREOF
    • C05FORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
    • C05F11/00Other organic fertilisers
    • C05F11/08Organic fertilisers containing added bacterial cultures, mycelia or the like
    • AHUMAN NECESSITIES
    • A01AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01NPRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • AHUMAN NECESSITIES
    • A01AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01NPRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/20Bacteria; Substances produced thereby or obtained therefrom
    • A01N63/22Bacillus
    • AHUMAN NECESSITIES
    • A01AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01NPRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/20Bacteria; Substances produced thereby or obtained therefrom
    • A01N63/27Pseudomonas
    • CCHEMISTRY; METALLURGY
    • C05FERTILISERS; MANUFACTURE THEREOF
    • C05DINORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C; FERTILISERS PRODUCING CARBON DIOXIDE
    • C05D3/00Calcareous fertilisers
    • C05D3/02Calcareous fertilisers from limestone, calcium carbonate, calcium hydrate, slaked lime, calcium oxide, waste calcium products

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  • Life Sciences & Earth Sciences (AREA)
  • Zoology (AREA)
  • Engineering & Computer Science (AREA)
  • Health & Medical Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Microbiology (AREA)
  • Pest Control & Pesticides (AREA)
  • Dentistry (AREA)
  • Virology (AREA)
  • Agronomy & Crop Science (AREA)
  • Plant Pathology (AREA)
  • Wood Science & Technology (AREA)
  • Environmental Sciences (AREA)
  • Biotechnology (AREA)
  • Organic Chemistry (AREA)
  • Biochemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Inorganic Chemistry (AREA)
  • Fertilizers (AREA)
  • Agricultural Chemicals And Associated Chemicals (AREA)

Abstract

Buluş, tarım sektöründe kullanılan bacillus subtilis (TV-17C), pseudomonas chlororapsis (MF-1), en az bir katı taşıyıcı, kalsiyum karbonat, magnezyum sülfat, demir sülfat, tween 20 ve yağ içeren katı taşıyıcılı bir mikrobiyal gübre ve biyopestisit formülasyonu ve üretim yöntemi ile ilgilidir. Buluş özellikle, bacillus subtilis (TV-17C), pseudomonas chlororapsis (MF-1), pomza, perlit, diatomit, kalsiyum karbonat, magnezyum sülfat, demir sülfat, tween 20, ayçiçek yağı, polivinilalkol, sitrik asit ve potasyum sorbat içeren katı taşıyıcılı bir mikrobiyal gübre ve biyopestisit formülasyonu ile ilgilidir.The invention is a microbial fertilizer and biopesticide formulation with solid carriers containing bacillus subtilis (TV-17C), pseudomonas chlororapsis (MF-1), at least one solid carrier, calcium carbonate, magnesium sulfate, ferrous sulfate, tween 20 and oil used in the agricultural sector and related to the production method. In particular, the invention is a solid carrier containing bacillus subtilis (TV-17C), pseudomonas chlororapsis (MF-1), pumice, perlite, diatomite, calcium carbonate, magnesium sulfate, ferrous sulfate, tween 20, sunflower oil, polyvinylalcohol, citric acid and potassium sorbate. relates to a microbial fertilizer and biopesticide formulation.

Description

TARIFNAME KATI TASIYICILI BIR MIKROBIYAL GUBRE VE BIYOPESTISIT FORMULASYONU Teknik Alan Bulus, tarim sektöründe kullanilan bacillus subtilis, pseudomonas chlororapsis, en az bir kati tasiyici, kalsiyum karbonat, magnezyum sülfat, demir sülfat, tween 20 ve yag içeren kati tasiyicili bir mikrobiyal gübre ve biyopestisit formülasyonu ve üretim yöntemi ile Bulus özellikle, bacillus subtilis, pseudomonas chlororapsis, pomza, perlit, diatomit, kalsiyum karbonat, magnezyum sülfat, demir sülfat, tween 20, ayçiçek yagi, polivinilalkol, sitrik asit ve potasyum sorbat içeren kati tasiyicili bir mikrobiyal gübre ve biyopestisit formülasyonu ile ilgilidir. Teknigin Bilinen Durumu Günümüzde pek çok ülkede bitki hastalik ve zararlilari ile mücadelede kimyasal pestisit, bitki gelisimini etkileyici ve toprak iyilestirici olarak pek çok kimyasal gübre kullanilmaktadir. Kimyasallarin dogaya ve insanliga vermis oldugu zararlar artik çok iyi bilinmekte ancak hala kullanilmasindan vazgeçilememektedir. Gelismis ülkelerde tarim sektöründe kimyasal pestisit ve gübre tüketimi azaltilmakta, hastalik-zararli mücadelesinde ve bitki beslenmesinde kullanilan biyolojik ürünlerin miktari arttirilmaktadir. Bu yüzden son yillarda çevreyle dost, toksik etki göstermeyen biyolojik ürünlerin kullanimina yönelik çalismalar hizla artmaktadir. Bu çalismalar isiginda tarimsal üretimde asiri kimyasallarin kullaniminin olumsuz yan etkilerini azaltmak veya bütünüyle ortadan kaldirmak için alternatif tarimsal üretim sistemleri gelistirilmistir. Tarimsal üretim sistemlerinde en çok üzerinde durulan konu; tarimda kullanilan kimyasallarin azaltilarak biyolojik yöntemlerle sorunlarin çözülmesidir. Bu sayede tarimsal üretimde sentetik ürünleri azaltan, organik tarim stratejileri önem kazanmistir. Böylece kimyasal mücadele yerine biyolojik mücadele, kimyasal gübre yerine organik ve mikrobiyal gübre yöntemleri gelistirilmistir ve mikrobiyal temelli biyoteknolojik yöntem ve ürünler alternatif olarak sunulmustur. Bu ürünlerin üretimi ve tarimda yapilan uygulamalari oldukça parlak ve gelecege isik tutan sonuçlar göstermektedir.Bu bulus, kimyasallara alternatif olarak gelistirilmis biyolojik bir üründür. Dünya genelinde üretilen biyolojik ürünler genel olarak sivi tasiyicili formülasyonlardir. Uretimi kolay olmasi nedeniyle tercih edilmektedir. Ancak tasinmasi, depolanmasi ve raf ömrü bakimindan eksik kalmaktadir. Ayrica kati tasiyici kullanilarak gelistirilen biyolojik ürünlerde kullanilan hammadelerin maliyeti ve toksik madde içermemesi de çok önemli unsurlardir. Literatürde, konu ile ilgili asagidaki basvuruya rastlanmistir: ile ilgilidir. Bu bulus Iignokitozan tabanli nanokompozitler ile belirli bakteri suslarina ait mikrobioatanin gübre ve biyopestisit amaçli kullanilacak bir bitki asisi ile ilgilidir. Bu bulus ile sürdürülebilir tarim alaninda kullanilmak üzere saksi, fide viyolleri, strafor ya da karton vb. fide malzemelerine dogrudan uygulanabilir bir kaplama olarak kullanilabilecek ait bitki yararlilarina iliskindir. Suda çözünebilen bu kompozit, yavas salinim ile bitkiyi özellikle genç dönemlerinde hastaliklara karsi korurken gübre etkisi ile büyüyüp gelismesini saglamaktadir. Bulusun sivi halde sprey olarak tohumlara, köke ya da topraga uygulanabilen hali kök çürükçüllügü gibi bitki zararlilarina karsi bitkinin direncine katki saglamaktadir. Bu bulus kullanim alani açisindan benzer olsa da, gelistirilen formülasyon içerigi bakimindan tamamen farklidir. Bu alanda yapilan çalismalara bir örnek olarak sunulmustur. Sonuç olarak yukarida anlatilan olumsuzluklardan dolayi ve mevcut çözümlerin konu hakkindaki yetersizligi nedeniyle ilgili teknik alanda bir gelistirme yapilmasi gerekli kilinmistir.Bulusun Kisa Açiklamasi Mevcut bulus, yukarida bahsedilen gereksinimleri karsilayan, tüm dezavantajlari ortadan kaldiran ve ilave bazi avantajlar getiren kati tasiyicili bir mikrobiyal gübre ve biyopestisit formülasyonu ile ilgilidir. Bulusun öncelikli amaci, birçok hastalik ve zararlinin önüne geçerek toprakta verim artisi saglamaktir.Bulusun bir amaci, bitki hastaliklarina karsi biyopestisit olarak, bitki gelisimine olumlu etkilerinden dolayi mikrobiyal gübre olarak ve topraktaki yararli etkilerinden dolayi toprak iyilestirici olarak kullanilan bir formülasyon gelistirmektir. Bulusun bir diger amaci, topraktaki maddelerin bitki tarafindan daha iyi alinabilmesini saglamaktir. Yukarida anlatilan amaçlarin yerine getirilmesi için bulus tarim sektöründe kullanilan kati tasiyicili bir mikrobiyal gübre ve biyopestisit formülasyonu olup, bacillus subtilis (TV-17C), pseudomonas chlororapsis (M F-1), en az bir kati tasiyici, kalsiyum karbonat, magnezyum sülfat, demir sülfat, tween 20 ve yag ihtiva etmektedir. Bulusun yapisal ve karakteristik özellikleri ve tüm avantajlari asagida yazilan detayli açiklama sayesinde daha net olarak anlasilacaktir ve bu nedenle degerlendirmenin de detayli açiklama göz önüne alinarak yapilmasi gerekmektedir.Bulusun Detayli Açiklamasi Bu detayli açiklamada bulus konusu kati tasiyicili bir mikrobiyal gübre ve biyopestisit formülasyonu sadece konunun daha iyi anlasilmasina yönelik olarak ve hiçbir sinirlayici etki olusturmayacak sekilde açiklanmaktadir. Bulus, tarim sektöründe kullanilan kati tasiyicili bir mikrobiyal gübre ve biyopestisit formülasyonudur. Bulus konusu kati tasiyicili bir mikrobiyal gübre ve biyopestisit formülasyonu; bacillus subtilis (TV-170), pseudomonas chlororapsis (MF-1), en az bir kati tasiyici, kalsiyum karbonat, magnezyum sülfat, demir sülfat, tween 20 ve yag içermektedir. Söz konusu formülasyonda kullanilan bakteriler bacillus subtilis (TV-170) olarak, pseudomonas chlororapsis ise (M F-1) olarak kodlanmistir. Bulus konusu formülasyon tercihen; kati tasiyici olarak pomza, perlit ve diatomit içermektedir. Kati tasiyici olarak pek çok alternatif bulunmaktadir. Toprak kaynakli tasiyicilar (torf, kömür ), bitkisel kaynakli tasiyicilar (kompostlar, hayvan gübresi, soya küspesi), cansiz materyaller (vermikülit, alginat) kati tasiyici olarak kullanilabilir.Bulus konusu formülasyon tercihen; ayçiçek yagi içermesidir. Yag olarak baska bitkisel yaglar (zeytinyagi, kanola yagi) da kullanilabilir. Bulus konusu formülasyon; ek olarak gidalarda kullanilabilir özellikte bir koruyucu içermektedir. Koruyucu olarak tercihen polivinilalkol, sitrik asit ve potasyum sorbat kullanilmaktadir. Bulus konusu kati tasiyicili bir mikrobiyal gübre ve biyopestisit formülasyonu; bacillus subtilis (TV-17C), pseudomonas chlororapsis (MF-1), pomza, perlit, diatomit, kalsiyum karbonat, magnezyum sülfat, demir sülfat, tween 20, ayçiçek yagi, polivinilalkol, sitrik asit ve potasyum sorbat içermektedir. Bulus konusu formülasyon tercihen; agirlikça % 15-40 bacillus subtilis (TV-WC), % 15-40 sorbat içermektedir. Söz konusu formülasyon 'özellikle tercihen; agirlikça ortalama % 22,6 bacillus subtilis (TV- diatomit, % 2,1 kalsiyum karbonat, % 1,6 magnezyum sülfat, % 2,6 demir sülfat, % 1,6 sorbat içermektedir. Bulus konusu kati tasiyicili bir mikrobiyal gübre ve biyopestisit formülasyonu üretim yöntemi asagidaki islem basamaklarini içermektedir: i. Kati tasiyicilarin bgütülmesi, ii. Kati tasiyicilar ve besin maddelerinin bir otoklavda sterilize edilmesi, iii. Sterilize edilen unsurlarin bir etüvde kurutulmasi, iv. MF-1 (P. chlororapsis) ve TV-1TC (B. subtilis) bakterilerinin kati ve sivi besiyerinde gelistirilmesi, v. MF-1 ve TV-17C bakterilerinin konsantrasyonu ayarlanarak sivi besiyerlerinde gelistirilmesi, vi. TV-1TC bakterisinden endospor üretilmesi, MF-1 bakterisinin steril kati tasiyicilar ile karistirilip daha fazla gelisim saglamasi için inkübasyona birakilmasi, TV-1TC bakterisi ve endosporlarinin steril kati tasiyicilar ile karistirilarak kurutmaya birakilmasi, MF-1 ve TV-17C içerikli kati tasiyicilarin, tween 20 ve yag ile karistirilmasi, Koruyucu maddelerin suda çözülüp, otoklavda sterilize edildikten sonra kati tasiyicili formülasyona homojen bir sekilde karistirilmasi. Kati tasiyicili formülasyonun bir etüvde kurutularak, paketlenmesi. Bulusun tercih edilen bir uygulamasina göre; (i) islem basamaginda kati tasiyicilar ortalama 50-60 mesh çapinda bir ögütücüde ögütülmektedir. Bulusun tercih edilen bir uygulamasina göre; (iii) islem basamaginda kati tasiyicilar ortalama 35-45' OC'de bir etüvde kurutulmaktadir. Bulusun tercih edilen bir uygulamasina göre; (vii) islem basamaginda MF-1 bakterisinin steril kati tasiyicilar ile karistirilip daha fazla gelisim saglamasi için ortalama 25-30°C'de 48 saat süresince inkübasyona birakilmaktadir. TV-1'IC bakterisi ise 30-37 0C'ye ayarli etüvde inkübasyona birakilabilir. Bulusun tercih edilen bir uygulamasina göre; (viii) islem basamaginda TV-17C bakterisi ve endosporlari steril kati tasiyicilar ile karistirilarak ortalama 40-55°C'de 24 saat süresince kurutmaya birakilmaktadir. Bulusun tercih edilen bir uygulamasina göre; (xi) islem basamaginda kati tasiyicili formülasyon ortalama 25-30°C'de 24 saat süresince bir etüvde kurutulmaktadir. Asagidaki Tablo-19de kati tasiyicili bir mikrobiyal gübre ve biyopestisit için örnek bir formülasyon gösterilmektedir: Agirlikça Agirlikça Içerik Tercih edilen Kullanilabilir Miktar (%) Miktar (%) Pseudomonas chlororapsis (M F-1 ) 22'6 15'40 Perlit 13,7 1-50 Diatomit 13,7 1-50 Kalsiyum karbonat 2,1 0,5-5 Magnezyum sülfat 1,6 0,5-5 Demir sülfat 2,6 0,5-5 Ayçiçek yagi 5,2 1-10 Polivinilalkol 0,5 0,01-5 Sitrik asit 0,05 0,01-2 Potasyum sorbat 0,05 0,01-2 Tablo-1: Bulus konusu örnek bir mikrobiyal gübre ve biyopestisit kompozisyonu Bulus konusu formülasyonun üretim yöntemi asagida ayrintili olarak açiklanmistir: Ana tasiyici maddeler 50-60 mesh çapinda ögütülüp kullanima hazirlanmistir. Kati tasiyicilar (pomza, perlit, diatomit) ve besin maddeleri (kalsiyum karbonat, magnezyum sülfat, demir sülfat) iki esit parçaya bölünüp cam kavanozlara koyulmustur. Otoklavda 121°Ctde 40 dakika süre ile steril edilmistir. Daha sonra kati tasiyicilar ve besin maddeleri 40°Crye ayarli etüvde 24 saat süreyle bekletilerek kurutulmustur. -80°C'de LB (Lurient Broth) ortaminda bulunan bakteri kültürleri oda sicakliginda çözüldükten sonra NA (Nutrient Agar) ortami bulunan petriye üç fazli çizim teknigi kullanilarak aktarilmis ve 27°C'de 1 gün inkübasyona birakilmistir. Inkübasyon sonrasi NB (Nutrient Broth) sivi besiyerine bakteri kültürlerinden bir öze dolusu aktarimi yapilmis ve 1 gün inkübasyona birakilmistir. Sivi besiyeri ortaminda gelisen bakteri kültürünün konsatrasyonu türbidimetrede Inkübasyon sonrasi optimum pH'lari ayarlanmis NB sivi besiyerine türbidimetrede 1x10B kobiml=ye ayarlanmis bakteri kültürlerinden 1 ml aktarim yapilmis ve MF-1 2 gün, TV-17C 1 gün optimum gelistikleri 27°C ve 35°C sicakliklarda çalkalayicili inkübatörde inkübasyona birakilmistir. Türbidimetrede 1x108 kob/ml'ye ayarlanmis, TV-17C bakterileri BSM (Bacillus Sporulation Medium) besiyeri ortamina inoküle edilmis ve 5 gün 35°Cide inkübasona birakilarak spor üretimi saglanmistir. MF-1 bakterisi türbidimetrede 1x108 kob/ml'ye ayarlanip kati tasiyicilar ve besin maddeleri bulunan cam kavanozlardan birine ilave edilip iyice karistirilmis ve sonrasinda 48 saat 28°C'de inkübasyona birakilmistir.- TV-1TC bakteri sporlari diger cam kavanoza ilave edilip 50°Cide 1 gün kurutmaya birakilmistir. - Islemler sonrasi cam kavanozlardaki maddeler birlestirilip tek bir kavanoza indirgenmis, Tween 20 ve ayçiçegi yagi formülasyona ilave edilmis, tekrar iyice karistirilarak tüm parti'ik'i'il yüzeylerinin yag ile kaplanmasi gerçeklestirilmistir. - Koruyucu maddeler suda çözülerek otoklavda steril edilmis kati formülasyona homojen bir sekilde karismasi saglanmistir. - Daha sonra formülasyon yeniden 30°C'ye ayarli etüvde 24 saat süreyle bekletilerek kurutulmustur. - Elde edilen formülasyon paketlenmistir. Bulusun 'L'iretim yöntemiyle ilgili dikkat edilmesi gereken noktalar asagida siralanmistir: - T'L'im çalisma steril sartlarda steril malzemelerle gerçeklestirilmelidir. - Sterilizasyon 121°C altinda yapilmamalidir ve zamanina dikkat edilmelidir. - Bakteriler optimum sicakliklarda (M F-1 için 27°C ve TV-1TC için 35°C) gelistirilmeli, inkübasyon sürelerine dikkat edilmelidir. - Kullanilan maddeler belirlenen siraya göre zamani gelince ilave edilmelidir. - Kullanilan koruyucu maddenin miktari asilmamalidir. - Karistirma adimlarinda homojen karistirmaya özen gösterilmelidir. - Kurutma islemlerinde belirlenen sicakliklar kullanilmalidir. Bulusta yer alan unsurlarin (bakteriler ve diger hammaddeler) bulusun isleyisine olan katkilari ve 'özellikleri asagida açiklanmistir: Bacillus subtilis (TV-17C); Bacillus subtilis bakterisi çubuk sekilli ve gram-pozitif özelliktedir. Mezofilik bir yapiya sahip olan B. subtilis 25-35°C sicaklik araliginda gelisir. Aerobik özelliktedir fakat yakin zamanda yapilan çalismalarda aneorobik ortamda da büyüdükleri görülmüstür. Stres altinda dayanikli endospor üretmektedir. Yani asidik, alkali, oksidatif veya ozmotik durumlar olustugunda endospor 'üreterek kendini korumaya alir. Bu sayede zor sartlarla bas edebilecek ve uygun ortam olustugunda tekrar gelisimine devam edebilir. Bacillus organizmalarinin ana yasam alanlari topraktir ve bitki uygulamalarinda kullanim alani yaygindir. B. subtilis difficidin, mikobakilin, polimiksin, subilin gibi çok sayida antibiyotik üretebilmektedir. Ayrica protein, pektin ve nisasta gibi polimerleri parçalayarak karbon ve nitrojen döngülerine katkida bulunmaktadir. Bacillus subtilis bakterileri kitinaz, kullanilan önemli enzimler üretmektedir. B. subtilis TV-17C izolati in vitro kosullarda Alternaria alternata, Penicillium digitatum, Scierotinia scierotiorum, P. ultimum, ve P. italicum'a karsi antifungal aktivitelerinin oldugu görülmüstür. Ayrica depo kosullarinda bu bakteri izolatlarinin mandalina meyvelerinde dogal inokulasyonlar ile olusabilecek hastalik siddetini de önemli ölçüde azalttiklari görülmüstür. Tekiner et al. (2019b)=nin yapmis oldugu diger bir çalismada B. subtilis TV- 170 izolatlarinin in vitro kosullarda hasat öncesi ayvada önemli kayiplara sebep olan Alternaria çürüklügüne sebep olan patojen fungusa karsi önemli antifungal etki gösterdikleri belirlenmistir. Colletotrichum gloesporoides'in in vitro kosullarda mikrobiyal antagonistlerle mücadele edilebilirliginin arastirildigi bir diger çalismada ise; B. subtilis TV- 170 izolatinin da bulundugu toplam 9 adet biyoajan bakteri izolati kullanilmis tüm mikrobiyal antagonistlerin in vitro sartlarda patojen fungusun gelisimini engelledigi belirlenmistir. Toplam 13 adet biyoajan bakteri ve T. harzianum türüne ait toplam 2 fungus izolatinin in vitro sartlarda A. solaniiye karsi etkililigi test edilmistir. B. subtilis TV-17C izolatlarinin bu patojene karsi biyolojik mücadelede biyokontrol ajani olarak kullanilabilecegi belirtilmistir. Tekiner et al. (2018b), narda Coniella granati=ye karsi in vitro sartlarda patojene karsi antagonistik aktivitelerini test etmis, arastirma sonucunda B. cereus TV-85D izolatinin %66 etkinlikle en etkili izolat oldugu bunu sirasiyla %64 etkinlikle B. substilis TV-17C ve %60 etkinlikle B. substilis TV-6F izolatinin takip ettigini belirtmistir.Kirmizi Iahanada S. sclerotiorum (Lib.) de Bary'ye karsi yürütülen bir diger biyolojik mücadele çalismasinda ise; B. subtilis TV-1 TC straininin hem in vitroda hem de in vivoda etkili oldugu belirtilmistir. Pseudomonas chlororapsis (MF-1); Pseudomonas chlororaphis; çubuk sekilli, spor olusturmayan bir veya birden fazla polar flagella içeren, gram negatif, aerobik hetetrof bir bakteridir. Bakterinin optimum gelistigi sicaklik 20-28°C olan mezofilik bakterinin en iyi gelistigi pH 6,3-7,5 araliklarinin oldugu belirtilmistir. P. chlororaphis bakterisi pyoverdin veya floresant içermektedir. Floresant, bakteri bitkiye baglandiginda bulundugu bitkide bulundugu toprak demir açisindan kisitli ise sari-yesil pigment olarak ortaya çikmaktadir. Tarimda P. chlororaphis hiyar, biber, domates ve diger birçok tarimsal üründe kök çürüklügüne neden olan fungal patojenlere karsi antibiyotik ürettigi bilinmektedir. Fenazin- 1-karboksilamid, 2-hidroksifenazin, pirrolnitrin, hidrojen siyanür, kitinazlar ve proteazlar dahil olmak üzere çesitli antimikrobiyal ikincil metabolitler üretebilirler. P. chlororaphis suslarinin, biyo gübreleme, fitostimülasyon ve biyo-kontrol için asilayicilar olarak kullanilabilen bitki büyümesini destekleyen etkili bakteriler oldugu bildirilmistir.Ayrica, ABD Çevre Koruma Ajansi (EPA)"ya göre P. chlororaphis'in insanlar, yaban hayati veya çevre için patojenik olmadigi düsünülmektedir. Antimikrobiyal aktiviteler ve hayvanlar ve çevreler Için düsük risk, bakteriyi P. chlororaphis'i tarimda yüksek potansiyele sahip biyolojik kontrol ajanlari haline getirmistir. Kapya biberde önemli kayiplara neden olan F. proliferatum'un mücadelesinde P. chlororaphis MF-1 izolatinin in vivo kosullarda %70 oraninda hastalik gelisimi üzerine etkili oldugu belirtilmistir. Bir diger çalismada; P. chlororaphis MF-1'in patlican fidelerinde büyük verim kayiplarina neden olan B. cinerea'ya karsi in vivo sartlarda etkili oldugu tespit edilmistir. Yine P. chlororaphis MF-1 izolatinin kullanildigi bir diger çalismada da havuçta Geotrichum candidum ile mücadelede bu bakterinin biyoajan olarak kullanilabilecegi belirtilmistir. Kati Tasiyicilar; Tasiyici, canli PGPB,nin fabrikadan sahaya teslimi için bir araçtir. Tasiyicilar ile olusturulacak herhangi bir biyoformülasyon olmadan, birçok gelecek vaat eden PGPB,nin pazara ulasmasi neredeyse imkansizdir. Biyoformülasyon gelistirilirken ana tasiyici olarak gözenekli birçok ürün kullanilmaktadir. Biyoformülasyonda kullanilacak tasiyicinin düsük maliyetli, çevreye zararsiz, kolay steril edilebilen, üretim esnasi ve uygulamada kullanilabilir, kolay ulasilabilir ve uzun süre depolanabilme özelligine sahip olmasi gerekmektedir Tasiyici istenilen zamanda, iyi fizyolojik durumda yeterli sayida canli bakteri hücresi saglama kapasitesine sahip olmalidir. Seçilen tasiyici madde fazla sayida hedef organizmanin gelismesini saglamalidir. Biyoformülasyonlar için kullanilan tasiyicilar, bakterileri hücre ölümlerinden korurlar ve yasam oranlarini yüksek tutarlar. Bu bulusta kullanilan kati tasiyicilar pomza, pelit, diatomittir. Her birinin kullanim oranlari bakterinin içerisinde yasamini sürdürmesi ve uzun süre canliligini devam ettirebilmesi için oldukça önemlidir. Yapilan çalismalarda tek tek kullaniminin kombine kullanima oranla verimsiz oldugu tespit edilmistir. Bu sebeple kombinasyon kullanilmasina karar verilmistir. Pomza: Pomza, açik renkli, bosluklu, süngerimsi, volkanik olaylar neticesinde olusmus, fiziksel ve kimyasal etkenlere karsi dayanikli, gözenekli, silisli volkanik bir kayaçtir. Pomza yaygin biçimde gaz boslukludur. Olusumu sirasinda, bünyedeki gazlarin, ani olarak bünyeyi terketmesi ve ani soguma nedeniyle, makro ölçekten mikro ölçege kadar sayisiz gözenek içerir. Gözenekler arasi genelde (özellikle mikro gözenekler) baglantisiz bosluklu oldugundan, geçirgenligi (permeabilite) düsük, isi yalitimi oldukça yüksektir.Biyoformülasyonda 50-60 mesh çapinda ögütülmüs asidik pomza kullanilmistir. Üretilen formülasyonda kati tasiyici amaciyla kullanilmistir. Perlit: Perlitin en önemli özelligi bünyesine aldigi suyu uzun süre tutmasidir. Perlit, tarim topraginin fiziksel özelliklerini artirici "substrat" maddesi olarak kullanildiginda topraktaki su kaybini azaltarak ortamin nemini korumakta, gözenekliligiyle topragin havalanmasini saglamakta, böylece bitkiler için en uygun üreme ve büyüme ortami olusturmaktadir. Bu özellikleriyle perlit bahçelerde, seracilikta, fide yetistirmede, organik ve kültür tariminda yaygin olarak kullanilmaktadir. Biyoformülasyonda genlestirilmis perlit 50-60 mesh çapinda ögütülüp kullanilmistir. Üretilen formülasyonda bakterilerin kati tasiyicisi amaciyla kullanilmistir. Diatomit: Diatomit, volkanik aktivitelerin fazla oldugu yörelerde bulunan tatli ve tuzlu su haznelerinde fotosentez olayinin yogun olarak gerçeklestigi sig) derinliklerde yasayan tek hücreli alg türü olan diatome iskeletlerinin yani sira kil, kum, volkanik kül ve diger organik kalintilardan olusan bir kayaçtir. Bu bulusta 50-60 mesh çapinda ögütülmüs diatomit kullanilmistir. Uretilen formülasyonda bakterilerin kati tasiyicisi amaciyla kullanilmistir. Besin Maddeleri; Organizmalarin enerji saglayabilmesi, hücresel komponentleri yapabilmesi, gelismesi, çogalmasi ve yasayabilmesi için beslenmesi ve bu nedenle de çesitli gida maddelerini almasi gereklidir. Bu maddelerin bir bölümü dogrudan ortamlardan saglanmasina karsin bir kismi da hücre içinde sentezlenir. Böylece yasam için gerekli olan mikro ve makro moleküller hazirlanir ve gerekli yerlerde kullanilir. Mikroorganizmalarin yapilari incelendiginde, kuru agirliklarinin %95'inden fazla bir kismini bazi temel elementlerin (karbon, oksijen, hidrojen, nitrojen, sülfür, fosfor, potasyum, kalsiyum, magnezyum ve demir) olusturdugu görülür. Bunlara, ayni zamanda, fazla gereksinim duyulur ve bulunduklari ortamdan fazla miktarlarda alinirlar. Bu maddelere, makro element (makro nütrient) adi da verilmektedir. Bunlardan ilk 6 tanesi (major elementler), protein, karbonhidrat, Iipid, nukleik asit, vs. yapisinda da yer almaktadirlar. Geri kalan 4 tanesi de (min'or elementler), hücre içinde katyon olarak kalirlar ve çesitli biyokimyasal olgularda görev alirlar. Kalsiyum karbonat: Kalsiyum karbonat, halk arasinda kireç tasi olarak bilinen bir tür kimyasal bilesiktir. Bilesik formülü CaCOa seklindedir. Formülasyonda bakteriler için besin Magnezyum sülfat: Magnezyum sülfat kimyasal formülü MgSO4 olan, magnezyum, kükürt ve oksijen ihtiva eden bir inorganik tuz. Formülasyonda bakteriler için besin Demir sülfat: Demir (II) sülfat ya da demir sülfatin bilesik formülü FeSOrtür. Formülasyonda bakteriler için besin maddesi olarak kullanilmistir. Ticari adi Tween 20 olan polisorbat 20, iyonik olmayan bir yüzey aktif maddedir. Formülasyonda emülgatör olarak kullanilmaktadir. Ayçiçek yagi; Bakterilerin kati tasiyicida immobilizasyonu amaciyla kullanilmaktadir. Ayçiçek yagi formülasyonda bulunan bakterilerin stabilize olmalarini saglamaktadir. Polivinilalkol: Suda çözünür, reçinemsi bir polimerdir. Toksik degildir. Formülasyonda baglayici ve kaplama ajani olarak kullanilmaktadir. Sitrik asit; Diger adiyla limon tuzu olarak bilinen sitrik asit antioksidan özelliginden dolayi formülasyonda raf 'Ömrünü uzatmak amaciyla kullanilmaktadir. Potasyum sorbat; Potasyum sorbat sorbik asidin potasyum tuzudur. Suda çok çözünür olan beyaz bir tuzdur. Oncelikle bir gida koruyucu olarak kullanilir. Vücut üzerine toksit etkisi yoktur. Maya, küf ve bakteriler üzerine inhibitör etkisi vardir. Antimikrobiyal etkisi daha çok küflerdeki dehidrogenaz enzim sistemi üzerine etkilidir. Bakterilere karsi etkisi maya ve küflere karsi etkisinden daha azdir. Bulusla ilgili olarak yapilan deneysel çalismalar ve sonuçlari asagida açiklanmistir: Bulus için kullanilacak bakterilerilerin 165 rDNA gen bölgeleri izole edilmis sekansa gönderilmis ve NCBI' da tanilamalari yapilmistir. Ayrica MIS ve BIOLOG sistemleri de tanilamalari desteklenmistir. Kati tasiyicili (pomza, perlit, diatomit) mikrobiyal gübre ve biyopestisit formülasyonu gelistirilerek hiyarda Fusarium kök ve gövde çürüklügü hastaligina sebep olan Fusarium oxysporum f.sp. radicis-cucumerinumia karsi biyoajan olarak kullanilabilirligi ve bitki gelisimine olumlu etkisi oldugu belirlenmistir. Kullanilan kati tasiyici materyaller formülasyonda kullanilan bakterilerin ortama kolay adaptasyonunda ve bitki gelisimi üzerine daha etkili bulunmustur. Kullanilan formülasyon içerigindeki bakterilerin toprak iyilestirmesi yaptigi da belirlenmistir. Kimyasal pestisite göre gelistirilen formülasyonun hastalik üzerine oldukça etkili oldugu bulunmustur. Asagidaki Tablo 2'ye göre K-11 formülasyonun hastalik siddeti 1,47 olarak, pozitif kontrol ilaç uygulamasinin hastalik siddeti, formülasyon uygulamalarindan daha yüksek olarak 2,41 degerinde bulunmustur. Bu da kimyasal ilaca karsilik olusturulan formülasyonun hastaliga karsi oldukça etkili oldugunu göstermektedir. Ayrica kullanilan bakterilerin in vitro ortamda hastaliga karsi gösterdikleri etkinlik konusunda da çalismalar yapilmistir. Uygulamalar Hastalik Siddeti Pozitif Kontrol Kontrol Uygulamalari Negatif Kontrol Formülasyon Uygulamalari K-11 (+ET 46) 1,47 Tablo 2. Formülasyonun bitkiye uygulanmasi sonucu hastalik siddeti sonuçlari Bulus konusu ürünün 18. ay raf ömrü sonuçlarina göre; oda sicakliginda 1 gr bakteri içerikli kati tasiyicili formülasyonda 3,2 x 106 kob/gr olarak, buzdolabi sicakliginda ise 2,3 x 106 kob/gr olarak bulunmustur. Yapilan çalismalarda bulusun, Fusarium kök ve gövde çürüklügü hastaligina sebep olan Fusarium oxysporum f.sp. radicis-cucumerinum'a karsi biyopestisit olarak oldukça basarili sonuçlar alinmistir. Ayrica kullanilan bakterilerin daha birçok hastaliga karsi etkili oldugu, bitki gelisimine toprak verimliligine olumlu etkileri oldugu tespit edilmistir.Bulusun faydalari asagida açiklanmistir; - Insan sagligi ve çevre kirliligi yönünden riskli olmayan, biyobozunur ve pestisite alternatif yerli formülasyonlar olusturulmustur. - Olusturulan bu formülasyonlarin, hiyarda Fusarium kök ve gövde çürüklügü hastaligina karsi alternatif kimyasal ilaç uygulamasina kiyasla oldukça etkili oldugu belirlenmistir. - Olusturulan formülasyonlarin antifungal etkinliklerinin yaninda, bitki beslenmesine ve bitki büyümesine de önemli katkilar sagladigi tespit edilmistir. - 18 aylik muhafaza süresi boyunca yapilan çalismalara göre oldukça basarili raf ömrü sonuçlari alinmistir. - Formülasyon içerigindeki bakteriler sayesinde toprakta bulunan minerallerin bitkiye alinabilecek forma getirilip bitkiye alinmasi saglanmis ve bu sayede bitki gelisimi desteklenmistir. - Formülasyon içerigindeki bakteriler toprak kökenli bakterilerdir ve daha önce bitkilerin kök bölgesinden izole edilmistir. Bu sebeple kati tasiyicili formülasyonun bitkiye uygulanmasi sonucu topraga kolaylikla adapte olmakta ve uzun süre yasamini devam ettirerek bitki gelisimi ve hastalik için etkisi uzun sürmektedir. - Formülasyon içerigindeki bakterilerin biyoteknolojik öneme sahip fitaz ve kitinaz enzimlerini salgiladiklari belirlenmistir. Bu sayede bitki gelisimi ve hastalik üzerine Yapilan çalismalarda bulusun, bitki hastaliklarinin yaninda bitki gelisim parametrelerine de etkili oldugu belirlenmistir. Bulus hem biyopestistist hem de mikrobiyal gübre olarak kullanilabilecektir. Kati tasiyici (pomza, perlit, diatomit) kullanilarak gelistirilmis formülasyon sivi formülasyonlara kiyasla çok az yer kaplamakta tasima ve ulasimda oldukça kolaylik saglamaktadir. Ayrica yapilan çalismalarda yüksek raf ömrü sebebiyle üretilen formülasyonlara göre büyük avantaj göstermektedir. Kullanilan bakteriler bulusçular tarafindan izole edilmistir.Oncelikle kimyasallara alternatif biyolojik bir ürün gelistirilmis olmasi doga ve insanlik için çok büyük 'önem arz etmekte ve kimyasallarin zararali etkilerinin 'Önüne geçmektedir.Günümüzde sürdürülebilir tarim uygulamalari önemini giderek arttirmaktadir. Bulus tarimda sürdürülebilirlik ve organik tarim faaliyetlerininin uygulamaya aktarilmasini desteklemektedir. Bulusun uygulamaya aktarilmasi neticesinde dünya genelindeki en büyük problemlerden biri olan kimyasal kullanimi azaltilacaktir. Sürdürülebilir tarim desteklenecektir. TR TR DESCRIPTION A MICROBIAL FERTILIZER AND BIOPESTICIDE FORMULATION WITH A SOLID CARRIER Technical Field The invention is a microbial fertilizer and biopesticide formulation with a solid carrier used in the agricultural sector, containing bacillus subtilis, pseudomonas chlororapsis, at least one solid carrier, calcium carbonate, magnesium sulfate, iron sulfate, tween 20 and oil. and the production method. In particular, the invention is a solid carrier microbial fertilizer and biopesticide formulation containing bacillus subtilis, pseudomonas chlororapsis, pumice, perlite, diatomite, calcium carbonate, magnesium sulfate, ferrous sulfate, tween 20, sunflower oil, polyvinyl alcohol, citric acid and potassium sorbate. It is related to. State of the Art Today, in many countries, many chemical fertilizers are used as chemical pesticides, plant growth enhancers and soil improvers in the fight against plant diseases and pests. The damage caused by chemicals to nature and humanity is now well known, but their use still cannot be abandoned. In developed countries, the consumption of chemical pesticides and fertilizers in the agricultural sector is reduced, and the amount of biological products used in disease-pest control and plant nutrition is increased. Therefore, in recent years, studies on the use of environmentally friendly, non-toxic biological products have been rapidly increasing. In the light of these studies, alternative agricultural production systems have been developed to reduce or completely eliminate the negative side effects of the use of excessive chemicals in agricultural production. The most emphasized issue in agricultural production systems is; The aim is to solve problems with biological methods by reducing the chemicals used in agriculture. In this way, organic farming strategies that reduce synthetic products in agricultural production have gained importance. Thus, biological control instead of chemical control, organic and microbial fertilizer methods instead of chemical fertilizer have been developed, and microbial-based biotechnological methods and products have been presented as alternatives. The production of these products and their applications in agriculture show very bright and future-proof results. This invention is a biological product developed as an alternative to chemicals. Biological products produced worldwide are generally liquid carrier formulations. It is preferred because it is easy to produce. However, it is lacking in terms of transportation, storage and shelf life. In addition, the cost and lack of toxic substances of the raw materials used in biological products developed using solid carriers are also very important factors. In the literature, the following reference has been found on the subject: It is related to. This invention is about a plant vaccine that will be used for fertilizer and biopesticide purposes using Ignochitosan-based nanocomposites and microbiota of certain bacterial strains. With this invention, pots, seedling trays, styrofoam or cardboard etc. can be used in sustainable agriculture. It refers to plant beneficials that can be used as a coating that can be applied directly to seedling materials. This water-soluble composite, with its slow release, protects the plant against diseases, especially in its young stages, and enables it to grow and develop with the effect of fertilizer. The liquid form of the invention, which can be applied to seeds, roots or soil as a spray, contributes to the plant's resistance against plant pests such as root rot. Although this invention is similar in terms of its usage area, the developed formulation is completely different in terms of its content. It is presented as an example of the work done in this field. As a result, due to the drawbacks described above and the inadequacy of existing solutions on the subject, it has become necessary to make a development in the relevant technical field. Brief Description of the Invention The present invention is a solid carrier microbial fertilizer and biopesticide formulation that meets the above-mentioned requirements, eliminates all disadvantages and brings some additional advantages. It is related to. The primary purpose of the invention is to increase soil productivity by preventing many diseases and pests. One purpose of the invention is to develop a formulation that can be used as a biopesticide against plant diseases, as a microbial fertilizer due to its positive effects on plant development, and as a soil improver due to its beneficial effects on the soil. Another purpose of the invention is to ensure that the substances in the soil can be better taken up by the plant. In order to fulfill the purposes described above, the invention is a microbial fertilizer and biopesticide formulation with solid carriers used in the agricultural sector, consisting of bacillus subtilis (TV-17C), pseudomonas chlororapsis (M F-1), at least one solid carrier, calcium carbonate, magnesium sulfate, iron. Contains sulfate, tween 20 and oil. The structural and characteristic features and all the advantages of the invention will be understood more clearly thanks to the detailed explanation written below, and therefore the evaluation should be made by taking the detailed explanation into consideration. Detailed Description of the Invention In this detailed explanation, the subject of the invention, a microbial fertilizer and biopesticide formulation with a solid carrier, will only be better understood. It is explained for the sake of understanding and in a way that does not create any limiting effect. The invention is a microbial fertilizer and biopesticide formulation with a solid carrier used in the agricultural sector. The subject of the invention is a microbial fertilizer and biopesticide formulation with a solid carrier; Contains bacillus subtilis (TV-170), pseudomonas chlororapsis (MF-1), at least one solid carrier, calcium carbonate, magnesium sulfate, iron sulfate, tween 20 and oil. The bacteria used in the formulation in question are coded as bacillus subtilis (TV-170) and pseudomonas chlororapsis as (M F-1). The formulation subject to the invention is preferably; It contains pumice, perlite and diatomite as solid carriers. There are many alternatives as solid carriers. Soil-based carriers (peat, coal), plant-based carriers (composts, animal manure, soybean meal), inanimate materials (vermiculite, alginate) can be used as solid carriers. The formulation of the invention is preferably; It contains sunflower oil. Other vegetable oils (olive oil, canola oil) can also be used as oil. The formulation subject to the invention; In addition, it contains a preservative that can be used in food. Polyvinylalcohol, citric acid and potassium sorbate are preferably used as preservatives. The subject of the invention is a microbial fertilizer and biopesticide formulation with a solid carrier; Contains bacillus subtilis (TV-17C), pseudomonas chlororapsis (MF-1), pumice, perlite, diatomite, calcium carbonate, magnesium sulfate, iron sulfate, tween 20, sunflower oil, polyvinylalcohol, citric acid and potassium sorbate. The formulation subject to the invention is preferably; It contains 15-40% bacillus subtilis (TV-WC) and 15-40% sorbate by weight. The formulation in question 'especially preferably; It contains an average of 22.6% bacillus subtilis (TV-diatomite, 2.1% calcium carbonate, 1.6% magnesium sulfate, 2.6% iron sulfate, 1.6% sorbate by weight. The subject of the invention is a solid carrier microbial fertilizer and biopesticide The formulation production method includes the following process steps: i. Grinding the solid carriers, ii. Sterilizing the solid carriers and nutrients in an autoclave, iii. Drying the sterilized elements in an oven, iv. MF-1 (P. chlororapsis) and TV-1TC ( B. subtilis) bacteria in solid and liquid media, v. MF-1 and TV-17C bacteria in liquid media by adjusting their concentration, vi. Production of endospore from TV-1TC bacteria, mixing MF-1 bacteria with sterile solid carriers to ensure further growth. Allowing for incubation, mixing TV-1TC bacteria and endospores with sterile solid carriers and allowing them to dry, mixing MF-1 and TV-17C containing solid carriers with tween 20 and oil, dissolving the preservatives in water and sterilizing them in the autoclave and then homogeneously adding them to the solid carrier formulation. mixing. Drying the solid carrier formulation in an oven and packaging it. According to a preferred embodiment of the invention; In the (i) process step, solid carriers are ground in a grinder with an average diameter of 50-60 mesh. According to a preferred embodiment of the invention; (iii) In the process step, solid carriers are dried in an oven at an average temperature of 35-45 °C. According to a preferred embodiment of the invention; (vii) In the process step, MF-1 bacteria are mixed with sterile solid carriers and incubated at an average of 25-30°C for 48 hours to ensure further development. TV-1'IC bacteria can be incubated in an oven set at 30-37 0C. According to a preferred embodiment of the invention; (viii) In the process step, TV-17C bacteria and endospores are mixed with sterile solid carriers and left to dry for 24 hours at an average of 40-55°C. According to a preferred embodiment of the invention; In the (xi) process step, the solid carrier formulation is dried in an oven at an average temperature of 25-30°C for 24 hours. An example formulation for a solid carrier microbial fertilizer and biopesticide is shown in Table 19 below: Content by Weight Preferred Usable Quantity (%) Quantity (%) Pseudomonas chlororapsis (M F-1 ) 22'6 15'40 Perlite 13.7 1 -50 Diatomite 13.7 1-50 Calcium carbonate 2.1 0.5-5 Magnesium sulfate 1.6 0.5-5 Iron sulfate 2.6 0.5-5 Sunflower oil 5.2 1-10 Polyvinyl alcohol 0, 5 0.01-5 Citric acid 0.05 0.01-2 Potassium sorbate 0.05 0.01-2 Table-1: An exemplary microbial fertilizer and biopesticide composition of the invention. The production method of the formulation of the invention is explained in detail below: Main The carrier materials were ground to a diameter of 50-60 mesh and prepared for use. Solid carriers (pumice, perlite, diatomite) and nutrients (calcium carbonate, magnesium sulfate, iron sulfate) were divided into two equal parts and placed in glass jars. It was sterilized in an autoclave at 121°C for 40 minutes. Then, the solid carriers and nutrients were dried by keeping them in an oven set at 40°C for 24 hours. After the bacterial cultures in LB (Lurient Broth) medium at -80°C were thawed at room temperature, they were transferred to a petri dish with NA (Nutrient Agar) medium using the three-phase drawing technique and incubated at 27°C for 1 day. After incubation, a loopful of bacterial cultures was transferred to NB (Nutrient Broth) liquid medium and left to incubate for 1 day. The concentration of the bacterial culture grown in the liquid medium was measured on the turbidimeter. After incubation, 1 ml of the bacterial cultures adjusted to 1x10B cobiml was transferred to the NB liquid medium, whose optimum pH was adjusted, and MF-1 was kept for 2 days, TV-17C for 1 day, where they developed optimally at 27°C and 35°. It was incubated in a shaker incubator at C temperatures. The turbidimeter was adjusted to 1x108 cfu/ml, TV-17C bacteria were inoculated into BSM (Bacillus Sporulation Medium) medium and incubated at 35°C for 5 days to ensure spore production. MF-1 bacteria was adjusted to 1x108 cfu/ml on the turbidimeter and added to one of the glass jars containing solid carriers and nutrients, mixed well and then incubated for 48 hours at 28°C. - TV-1TC bacterial spores were added to the other glass jar and heated at 50°C. Cide was left to dry for 1 day. - After the processes, the ingredients in the glass jars were combined and reduced to a single jar, Tween 20 and sunflower oil were added to the formulation, mixed thoroughly again and all the particles' surfaces were covered with oil. - Preservatives were dissolved in water and mixed homogeneously into the solid formulation that was sterilized in an autoclave. -Then, the formulation was dried again by keeping it in the oven set at 30°C for 24 hours. - The resulting formulation is packaged. The points to be considered regarding the 'L' production method of the invention are listed below: - The 'L' work must be carried out under sterile conditions with sterile materials. - Sterilization should not be done below 121°C and time should be taken into consideration. - Bacteria should be grown at optimum temperatures (27°C for M F-1 and 35°C for TV-1TC), and attention should be paid to incubation periods. - The ingredients used should be added when the time comes, in the specified order. - The amount of preservative used should not be exceeded. - Care should be taken to ensure homogeneous mixing during the mixing steps. - Specified temperatures should be used in drying processes. The contributions and properties of the elements (bacteria and other raw materials) included in the invention to the operation of the invention are explained below: Bacillus subtilis (TV-17C); Bacillus subtilis bacteria is rod-shaped and gram-positive. B. subtilis, which has a mesophilic structure, grows in the temperature range of 25-35°C. They are aerobic, but recent studies have shown that they also grow in an anaerobic environment. It produces durable endospore under stress. In other words, when acidic, alkaline, oxidative or osmotic conditions occur, it protects itself by producing endospore. In this way, it can cope with difficult conditions and continue its development when the appropriate environment is created. The main habitat of Bacillus organisms is soil and its use in plant applications is widespread. B. subtilis can produce many antibiotics such as difficidin, mycobacilin, polymyxin, subilin. It also contributes to carbon and nitrogen cycles by breaking down polymers such as protein, pectin and starch. Bacillus subtilis bacteria produce chitinase, important enzymes used in B. subtilis TV-17C isolate has been shown to have antifungal activities against Alternaria alternata, Penicillium digitatum, Scierotinia scierotiorum, P. ultimum, and P. italicum under in vitro conditions. In addition, it has been observed that these bacterial isolates significantly reduce the severity of disease that may occur with natural inoculations in tangerine fruits under storage conditions. Tekiner et al. (2019b) found that B. subtilis TV-170 isolates showed significant antifungal effects against the pathogenic fungus that causes Alternaria rot, which causes significant losses in quince before harvest, under in vitro conditions. In another study, the ability of Colletotrichum gloesporoides to be combated with microbial antagonists under in vitro conditions was investigated; A total of 9 bioagent bacterial isolates, including B. subtilis TV-170 isolate, were used and it was determined that all microbial antagonists prevented the growth of pathogenic fungi under in vitro conditions. The effectiveness of a total of 13 bioagent bacteria and a total of 2 fungal isolates belonging to the T. harzianum species was tested against A. solanii under in vitro conditions. It has been stated that B. subtilis TV-17C isolates can be used as biocontrol agents in biological control against this pathogen. Tekiner et al. (2018b) tested the antagonistic activities of pomegranate against Coniella granati in in vitro conditions against the pathogen, and as a result of the research, B. cereus TV-85D isolate was the most effective isolate with 66% effectiveness, followed by B. substilis TV-17C and 60% effectiveness, respectively. stated that B. substilis TV-6F isolate was the most effective. In another biological control study conducted against S. sclerotiorum (Lib.) de Bary in Red Cabbage; It has been stated that B. subtilis TV-1 TC strain is effective both in vitro and in vivo. Pseudomonas chlororapsis (MF-1); Pseudomonas chlororaphis; It is a rod-shaped, gram-negative, aerobic heterotrophic bacterium that does not form spores and contains one or more polar flagella. It has been stated that the optimum temperature for bacteria to grow is 20-28°C, and the pH range for mesophilic bacteria to grow best is 6.3-7.5. P. chlororaphis bacteria contain pyoverdin or fluorescent. Fluorescent appears as a yellow-green pigment when the bacterium attaches to the plant and the soil in which it is found is limited in terms of iron. In agriculture, P. chlororaphis is known to produce antibiotics against fungal pathogens that cause root rot in cucumbers, peppers, tomatoes and many other agricultural products. They can produce a variety of antimicrobial secondary metabolites, including phenazine-1-carboxylamide, 2-hydroxyphenazine, pyrrolnitrine, hydrogen cyanide, chitinases, and proteases. P. chlororaphis strains have been reported to be effective plant growth-promoting bacteria that can be used as inoculants for biofertilization, phytostimulation, and biocontrol. Additionally, according to the US Environmental Protection Agency (EPA), P. chlororaphis is not pathogenic to humans, wildlife, or the environment. Antimicrobial activities and low risk to animals and environments have made the bacteria P. chlororaphis high potential biological control agents in agriculture. P. chlororaphis MF-1 isolate was used in vivo in the fight against F. proliferatum, which causes significant losses in capia pepper. It has been stated that it is effective on disease development at a rate of 70% under conditions. In another study, it was determined that P. chlororaphis MF-1 is effective against B. cinerea, which causes great yield losses in eggplant seedlings, under in vivo conditions. Again, P. chlororaphis MF- In another study using 1 isolate, it was stated that this bacterium could be used as a bioagent in the fight against Geotrichum candidum in carrots. Solid Carriers; The carrier is a vehicle for the delivery of live PGPB from the factory to the field. Without any bioformulation with carriers, it is almost impossible for many promising PGPBs to reach the market. While developing bioformulations, many porous products are used as the main carrier. The carrier to be used in bioformulation must be low-cost, harmless to the environment, easily sterilized, usable during production and in practice, easily accessible and able to be stored for a long time. The carrier must have the capacity to provide a sufficient number of live bacterial cells in a good physiological state at the desired time. The selected carrier substance must ensure the growth of a large number of target organisms. Carriers used for bioformulations protect bacteria from cell death and keep survival rates high. The solid carriers used in this invention are pumice, pelite and diatomite. The usage rates of each are very important for the bacteria to survive within it and maintain its vitality for a long time. Studies have found that using them individually is inefficient compared to combined use. For this reason, it was decided to use a combination. Pumice: Pumice is a light-colored, porous, spongy, porous, siliceous volcanic rock formed as a result of volcanic events, resistant to physical and chemical factors. Pumice is extensively gas-filled. It contains countless pores, from macro to micro scale, due to the sudden departure of gases from the body and sudden cooling during its formation. Since the pores are generally unconnected (especially micro pores), its permeability is low and its thermal insulation is quite high. 50-60 mesh diameter ground acidic pumice was used in the bioformulation. It was used as a solid carrier in the produced formulation. Perlite: The most important feature of perlite is that it retains the water it absorbs for a long time. When perlite is used as a "substrate" material that increases the physical properties of agricultural soil, it preserves the humidity of the environment by reducing water loss in the soil, provides ventilation of the soil with its porosity, thus creating the most suitable breeding and growth environment for plants. With these properties, perlite is widely used in gardens, greenhouses, seedling growing, organic and cultural agriculture. Expanded perlite was ground and used in 50-60 mesh diameter in bioformulation. It was used as a solid carrier of bacteria in the produced formulation. Diatomite: Diatomite is a rock composed of diatom skeletons, which are a type of single-celled algae that live at shallow depths where photosynthesis occurs intensively, in fresh and salt water reservoirs in regions with high volcanic activity, as well as clay, sand, volcanic ash and other organic residues. In this invention, ground diatomite with a diameter of 50-60 mesh was used. It was used as a solid carrier of bacteria in the produced formulation. Nutrients; Organisms need to be fed and therefore receive various nutrients in order to provide energy, make cellular components, develop, reproduce and survive. Although some of these substances are obtained directly from the environment, some are synthesized within the cell. Thus, the micro and macro molecules necessary for life are prepared and used where necessary. When the structures of microorganisms are examined, it is seen that more than 95% of their dry weight consists of some basic elements (carbon, oxygen, hydrogen, nitrogen, sulfur, phosphorus, potassium, calcium, magnesium and iron). At the same time, they are needed in large quantities and are taken in large quantities from the environment they are in. These substances are also called macro elements (macro nutrients). The first 6 of these (major elements) are protein, carbohydrate, lipid, nucleic acid, etc. They are also included in its structure. The remaining 4 (minor elements) remain as cations within the cell and take part in various biochemical phenomena. Calcium carbonate: Calcium carbonate is a type of chemical compound popularly known as limestone. The compound formula is CaCOa. Nutrient for bacteria in the formulation Magnesium sulfate: Magnesium sulfate is an inorganic salt containing magnesium, sulfur and oxygen, with the chemical formula MgSO4. Nutrient for bacteria in the formulation Ferrous sulfate: Iron (II) sulfate or the compound formula of ferrous sulfate is FeSO. It was used as a nutrient for bacteria in the formulation. Polysorbate 20, whose trade name is Tween 20, is a non-ionic surfactant. It is used as an emulsifier in the formulation. Sunflower oil; It is used for the immobilization of bacteria on solid carriers. Sunflower oil ensures the stabilization of the bacteria in the formulation. Polyvinylalcohol: It is a water-soluble, resinous polymer. It is not toxic. It is used as a binding and coating agent in the formulation. Citric acid; Citric acid, also known as lemon salt, is used in the formulation to extend shelf life due to its antioxidant properties. Potassium sorbate; Potassium sorbate is the potassium salt of sorbic acid. It is a white salt that is very soluble in water. It is primarily used as a food preservative. It has no toxic effect on the body. It has an inhibitory effect on yeast, mold and bacteria. Its antimicrobial effect is mostly effective on the dehydrogenase enzyme system in molds. Its effect against bacteria is less than its effect against yeast and moulds. Experimental studies and their results regarding the invention are explained below: 165 rDNA gene regions of the bacteria to be used for the invention were isolated, sent to sequence, and identified at NCBI. Additionally, MIS and BIOLOG systems are also supported for diagnosis. Fusarium oxysporum f.sp., which causes Fusarium root and stem rot disease in cucumber, was developed by developing a microbial fertilizer and biopesticide formulation with solid carriers (pumice, perlite, diatomite). It has been determined that it can be used as a bioagent against radicis-cucumerinumia and has a positive effect on plant development. The solid carrier materials used were found to be more effective in the easy adaptation of the bacteria used in the formulation to the environment and on plant development. It has also been determined that the bacteria in the formulation used improve the soil. It has been found that the formulation developed based on chemical pesticide is very effective on the disease. According to Table 2 below, the disease severity of the K-11 formulation was found to be 1.47, and the disease severity of the positive control drug application was found to be 2.41, higher than the formulation applications. This shows that the formulation created in response to the chemical drug is quite effective against the disease. Studies have also been conducted on the effectiveness of the bacteria used against the disease in vitro. Applications Disease Severity Positive Control Control Applications Negative Control Formulation Applications K-11 (+ET 46) 1.47 Table 2. Disease severity results as a result of application of the formulation to the plant. According to the 18th month shelf life results of the product of the invention; It was found to be 3.2 x 106 cfu/gr in the solid carrier formulation containing 1 g of bacteria at room temperature, and 2.3 x 106 cfu/gr at refrigerator temperature. Studies have shown that Fusarium oxysporum f.sp., which causes Fusarium root and stem rot disease. Very successful results have been obtained as a biopesticide against radicis-cucumerinum. It has also been determined that the bacteria used are effective against many diseases and have positive effects on plant growth and soil fertility. The benefits of the invention are explained below; - Local formulations that are not risky to human health and environmental pollution, are biodegradable and alternative to pesticides have been created. - It has been determined that these formulations are quite effective against Fusarium root and stem rot disease in cucumber, compared to alternative chemical pesticide application. - It has been determined that the created formulations, in addition to their antifungal activities, also make significant contributions to plant nutrition and plant growth. - According to the studies conducted during the 18-month storage period, very successful shelf life results were obtained. - Thanks to the bacteria in the formulation, the minerals in the soil are brought to a form that can be taken into the plant and thus plant development is supported. - The bacteria in the formulation are soil-borne bacteria and have been previously isolated from the root zone of plants. For this reason, as a result of the application of the solid carrier formulation to the plant, it easily adapts to the soil and survives for a long time, providing a long-lasting effect on plant development and disease. - It has been determined that the bacteria in the formulation secrete phytase and chitinase enzymes, which are of biotechnological importance. In this way, in studies on plant development and disease, it has been determined that the invention is effective on plant development parameters as well as plant diseases. The invention can be used both as a biopesticide and as a microbial fertilizer. The formulation developed using solid carriers (pumice, perlite, diatomite) takes up very little space compared to liquid formulations and provides great convenience in transportation and transportation. Additionally, studies have shown that it has a great advantage over produced formulations due to its high shelf life. The bacteria used were isolated by the inventors. First of all, the development of a biological product alternative to chemicals is of great importance for nature and humanity and prevents the harmful effects of chemicals. Nowadays, sustainable agricultural practices are increasingly gaining importance. The invention supports sustainability in agriculture and the implementation of organic farming activities. As a result of putting the invention into practice, chemical use, which is one of the biggest problems worldwide, will be reduced. Sustainable agriculture will be supported.TR TR

Claims (1)

1.ISTEMLER .Tarim sektöründe kullanilan kati tasiyicili bir mikrobiyal gübre ve biyopestisit formülasyonu olup, özelligi; bacillus subtilis (TV-17C), pseudomonas chlororapsis (MF-1), en az bir kati tasiyici, kalsiyum karbonat, magnezyum sülfat, demir sülfat, tween 20 ve yag içermesidir. . istem 1'e uygun kati tasiyicili bir mikrobiyal gübre ve biyopestisit formülasyonu olup, özelligi; kati tasiyici olarak pomza, perlit ve diatomit içermesidir. . istem 1'e uygun kati tasiyicili bir mikrobiyal gübre ve biyopestisit formülasyonu olup, özelligi; ayçiçek yagi içermesidir. . istem 1'e uygun kati tasiyicili bir mikrobiyal gübre ve biyopestisit formülasyonu olup, özelligi; ek olarak gidalarda kullanilabilir özellikte bir koruyucu içermesidir. . istem 4'e uygun kati tasiyicili bir mikrobiyal gübre ve biyopestisit formülasyonu olup, özelligi; koruyucu olarak polivinilalkol, sitrik asit ve potasyum sorbat içermesidir. . istem 1'e uygun kati tasiyicili bir mikrobiyal gübre ve biyopestisit formülasyonu olup, özelligi; bacillus subtilis (TV-17C), pseudomonas chlororapsis (MF-1), pomza, perlit, diatomit, kalsiyum karbonat, magnezyum sülfat, demir sülfat, tween 20, ayçiçek yagi, polivinilalkol, sitrik asit ve potasyum sorbat içermesidir. . istem 6rya uygun kati tasiyicili bir mikrobiyal gübre ve biyopestisit formülasyonu olup, içermesidir. . istem ?iye uygun kati tasiyicili bir mikrobiyal gübre ve biyopestisit formülasyonu olup, özelligi; agirlikça ortalama % 22,6 bacillus subtilis (TV-17C), % 22,6 pseudomonas yagi, % 0,5 polivinilalkol, % 0,05 sitrik asit ve % 0,05 potasyum sorbat içermesidir. .Tarim sektöründe kullanilan kati tasiyicili bir mikrobiyal gübre ve biyopestisit formülasyonu üretim yöntemi olup, özelligi; asagidaki islem basamaklarini içermesidir: i. Kati tasiyicilarin ögütülmesi, ii. Kati tasiyicilar ve besin maddelerinin bir otoklavda sterilize edilmesi, iii. Sterilize edilen unsurlarin bir etüvde kurutulmasi, iv. MF-1 (P. chlororapsis) ve TV-l'z'C (B. subtilis) bakterilerinin kati ve sivi besiyerinde gelistirilmesi, v. MF-l ve TV-1TC bakterilerinin konsantrasyonu ayarlanarak sivi besiyerlerinde gelistirilmesi, vi. TV-17C bakterisinden endospor üretilmesi, vii. MF-1 bakterisinin steril kati tasiyicilar ile karistirilip daha fazla gelisim saglamasi için inkübasyona birakilmasi, viii.TV-1TC bakterisi ve endosporlarinin steril kati tasiyicilar ile karistirilarak kurutmaya birakilmasi, ix. MF-1 ve TV-17C içerikli kati tasiyicilarin, tween 20 ve yag ile karistirilmasi, x. Koruyucu maddelerin suda çözülüp, otoklavda sterilize edildikten sonra kati tasiyicili formülasyona homojen bir sekilde karistirilmasi, xi. Kati tasiyicili formülasyonun bir etüvde kurutularak, paketlenmesi. Istem 9ia uygun kati tasiyicili bir mikrobiyal gübre ve biyopestisit formülasyonu üretim yöntemi olup, özelligi; (i) islem basamaginda kati tasiyicilarin ortalama 50-60 mesh çapinda ögütülmesidir. Istem 9'a uygun kati tasiyicili bir mikrobiyal gübre ve biyopestisit formülasyonu üretim yöntemi olup, özelligi; (iii) islem basamaginda kati tasiyicilarin ortalama 35-45; °Cide bir etüvde kurutulmasidir. Istem 9'a uygun kati tasiyicili bir mikrobiyal gübre ve biyopestisit formülasyonu üretim yöntemi olup, özelligi; (vii) islem basamaginda MF-1 bakterisinin steril kati tasiyicilar ile karistirilip daha fazla gelisim saglamasi için ortalama 25-30°C=de 48 saat süresince inkübasyona birakilmasidir. 13. Istem 9'a uygun kati tasiyicili bir mikrobiyal gübre ve biyopestisit formülasyonu üretim yöntemi olup, özelligi; (viii) islem basamaginda TV-17C bakterisi ve endosporlarinin steril kati tasiyicilar ile karistirilarak ortalama 40-55°C=de 24 saat süresince kurutmaya birakilmasidir. 5 14. Istem 93 uygun kati tasiyicili bir mikrobiyal gübre ve biyopestisit formülasyonu üretim yöntemi olup, özelligi; (xi) islem basamaginda kati tasiyicili formülasyonun ortalama 25-30°C!de 24 saat süresince bir etüvde kurutulmasidir. TR TR1. CLAIMS: It is a solid carrier microbial fertilizer and biopesticide formulation used in the agricultural sector, and its feature is; It contains bacillus subtilis (TV-17C), pseudomonas chlororapsis (MF-1), at least one solid carrier, calcium carbonate, magnesium sulfate, iron sulfate, tween 20 and oil. . It is a solid carrier microbial fertilizer and biopesticide formulation in accordance with claim 1, and its feature is; It contains pumice, perlite and diatomite as solid carriers. . It is a solid carrier microbial fertilizer and biopesticide formulation in accordance with claim 1, and its feature is; It contains sunflower oil. . It is a solid carrier microbial fertilizer and biopesticide formulation in accordance with claim 1, and its feature is; In addition, it contains a preservative that can be used in food. . It is a solid carrier microbial fertilizer and biopesticide formulation in accordance with claim 4, and its feature is; It contains polyvinyl alcohol, citric acid and potassium sorbate as preservatives. . It is a solid carrier microbial fertilizer and biopesticide formulation in accordance with claim 1, and its feature is; It contains bacillus subtilis (TV-17C), pseudomonas chlororapsis (MF-1), pumice, perlite, diatomite, calcium carbonate, magnesium sulfate, iron sulfate, tween 20, sunflower oil, polyvinylalcohol, citric acid and potassium sorbate. . It is a microbial fertilizer and biopesticide formulation with a solid carrier in accordance with claim 6 and contains it. . It is a microbial fertilizer and biopesticide formulation with solid carrier in accordance with the claim and its feature is; It contains an average of 22.6% bacillus subtilis (TV-17C), 22.6% pseudomonas oil, 0.5% polyvinyl alcohol, 0.05% citric acid and 0.05% potassium sorbate by weight. It is a solid carrier microbial fertilizer and biopesticide formulation production method used in the agricultural sector, and its feature is; It includes the following process steps: i. Grinding of solid carriers, ii. Sterilizing solid carriers and nutrients in an autoclave, iii. Drying the sterilized items in an oven, iv. Growth of MF-1 (P. chlororapsis) and TV-1'z'C (B. subtilis) bacteria in solid and liquid media, v. Development of MF-1 and TV-1TC bacteria in liquid media by adjusting their concentration, vi. Production of endospore from TV-17C bacteria, vii. Mixing MF-1 bacteria with sterile solid carriers and allowing them to incubate to ensure further development, viii. TV-1TC bacteria and their endospores mixing them with sterile solid carriers and allowing them to dry, ix. Mixing of solid carriers containing MF-1 and TV-17C with tween 20 and oil, x. Dissolving the preservatives in water, sterilizing them in an autoclave, and then mixing them homogeneously into the solid carrier formulation, xi. Drying the solid carrier formulation in an oven and packaging it. It is a solid carrier microbial fertilizer and biopesticide formulation production method in accordance with claim 9, and its feature is; (i) In the process step, the solid carriers are ground to an average diameter of 50-60 mesh. It is a solid carrier microbial fertilizer and biopesticide formulation production method in accordance with claim 9, and its feature is; (iii) average of 35-45 solid carriers in the process step; °It means drying the skin in an oven. It is a solid carrier microbial fertilizer and biopesticide formulation production method in accordance with claim 9, and its feature is; (vii) In the process step, MF-1 bacteria are mixed with sterile solid carriers and left to incubate for 48 hours at an average of 25-30°C to ensure further development. 13. It is a solid carrier microbial fertilizer and biopesticide formulation production method according to claim 9, and its feature is; (viii) In the process step, TV-17C bacteria and their endospores are mixed with sterile solid carriers and left to dry for 24 hours at an average of 40-55°C. 5 14. Claim 93 is a microbial fertilizer and biopesticide formulation production method with a suitable solid carrier, and its feature is; (xi) In the process step, the solid carrier formulation is dried in an oven at an average temperature of 25-30°C for 24 hours. TR TR
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