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658732.Pdf (7.033Mb) ANKARA ÜNİVERSİTESİ FENBİLİMLERİ ENSTİTÜSÜ DOKTORA TEZİ ANKARA İLİ TARIM DIŞI ALANLARDA BULUNAN ENTOMOPATOJEN NEMATODLARIN VE SİMBİYOTİK BAKTERİLERİNİN TANIMLANMASI VE ETKİNLİKLERİNİN BELİRLENMESİ ESENGÜL ÖZDEMİR BİTKİ KORUMA ANABİLİM DALI ANKARA 2021 Her hakkı saklıdır ÖZET DOKTORA TEZİ ANKARA İLİ TARIM DIŞI ALANLARDA BULUNAN ENTOMOPATOJEN NEMATODLARIN VE SİMBİYOTİK BAKTERİLERİNİN TANIMLANMASI VE ETKİNLİKLERİNİN BELİRLENMESİ Esengül ÖZDEMİR Ankara Üniversitesi Fen Bilimleri Enstitüsü Bitki Koruma Anabilim Dalı Danışman: Prof. Dr. Selma ÜLGENTÜRK Bu çalışma, Ankara ili tarım dışı alanlarda bulunan entomopatojen nematod (EPN) faunasının ve bu nematodlarla simbiyotik yaşayan bakterilerin belirlenmesi amacıyla 2017-2020 yıllarında gerçekleştirilmiştir. Çalışma alanı, Ankara ili sınırları içerisinde bulunan 9 adet Tabiat Parkı, 1 adet Milli Park, 3 adet orman ve 10 adet doğal mağaradan oluşmuştur. Çalışma kapsamında, belirtilen alanlardan toplam 636 toprak örneği alınmıştır. Bu toprak örneklerinin 31 tanesinden (% 5.66) G. mellonella tuzak yöntemi kullanılarak, 6 farklı EPN türü elde edilmiştir. Elde edilen EPN izolatlarının yapılan morfometrik ölçümler ve ITS rDNA bölgesinin sekans analizi sonucunda tür teşhisleri yapılmıştır. Elde edilmiş 31 izolattan, Steinernema cinsine ait olan 14 tanesinin Steinernema feltiae, 5 tanesinin Steinernema affine, 6 tanesinin Steinernema litorale, 3 tanesinin S. weiseri ve 1 tanesinin ise Steinernema bicornutum türleri olduğu tespit edilmiştir. Heterorhabditis cinsine ait olan 2 izolatın ise Heterorhabditis bacteriophora olduğu belirlenmiştir. Bu nematodlardan bakteri izolasyonları yapılmış ve 16S rRNA bölgeleri çoğaltılarak sekans analizleri yapılmıştır. Çalışmanın sonucunda S. feltiae, S. affine, S. litorale ve S. weiseri türlerinden elde edilen simbiyotik bakterileri Xenorhabdus bovienii, S. bicornutum türünün simbiyotik bakterisi Xenorhabdus budapestensis, H. bacteriophora türü iki izolatın simbiyotik bakterileri ise Photorhabdus luminescens subsp. kayaii olarak tespit edilmiştir. Tez çalışması kapsamında, EPN’lerin Galleria mellonella larvaları üzerindeki biyolojik etkinlikleri de değerlendirilmiştir. Elde edilen sonuçlar, test edilen EPN izolatlarının G. mellonella son dönem larvalarına karşı biyolojik mücadele potansiyellerinin yüksek olduğunu göstermiştir. Şubat 2021, 234 Sayfa Anahtar Kelimeler: Steinernema, Heterorhabditis, Xenorhabdus, Photorhabdus, entomopatojen nematod, simbiyotik bakteri, biyolojik mücadele ii ABSTRACT Ph. D. Thesis IDENTIFICATION OF INDIGENOUS ENTOMOPATHOGENIC NEMATODES AND THEIR SYMBIOTIC BACTERIA AND DETERMINING THEIR EFFICIENCY IN NON- AGRİCULTURAL LANDS OF ANKARA Esengül ÖZDEMİR Ankara University Graduate School of Natural and Applied Sciences Department of Plant Protection Supervisor: Prof. Dr. Selma ÜLGENTÜRK This study was carried out in 2017-2020 to determine the entomopathogenic nematode fauna and the symbiotic bacteria of these nematodes in non-agricultural areas of Ankara. The study area consists of 9 Nature Parks, 1 National Park, 3 forests and 8 natural caves within the borders of Ankara province with altitudes from 850 meters to 1540 meters. In the study, a total of 636 soil samples were taken from the specified areas. Six different EPN species were obtained from 36 of these soil samples by using Galleria mellonella trap method. The rate of obtaining entomopathogenic nematodes from soil samples was determined as 5.66%. 34 of the isolates obtained belong to the genus Steinernema and 2 isolates belong to the genus Heterorhabditis. Species of the obtained EPN isolates were identified as a result of morphometric measurements and sequence analysis of the ITS rDNA region. Of the 31 isolates obtained, 14 were Steinernema feltiae, 5 were Steinernema affine, 6 were Steinernema litorale, 3 were S. weiseri and 1 was Steinernema bicornutum. The 2 isolates belonging to the genus Heterorhabditis were determined to be Heterorhabditis bacteriophora. Bacterial isolations were performed from these EPNs and sequence analyses were performed by amplifying 16S rRNA region. As a result of the study, the symbiotic bacteria Xenorhabdus bovienii obtained from S. feltiae, S. affine, S. litorale and S. weiseri species, the symbiotic bacterium of H. bacteriophora isolates were identified as Photorhabdus luminescens subsp. kayaii. Within the scope of the thesis, the biological activities of EPNs on G. mellonella larvae were also evaluated. The results showed that the tested EPN isolates have high biological control potential against G. mellonella last instar larvae. February 2021, 234 Pages Keywords: Steinernema, Heterorhabditis, Xenorhabdus, Photorhabdus, entomopathogenic nematod, simbiotic bacteria, biological control iii TEŞEKKÜR Ankara ili tarım dışı alanlarda bulunan entomopatojen nematodların ve simbiyotik bakterilerinin tanımlanması ve etkinliklerinin belirlenmesi amacıyla yürüttüğüm çalışmalar boyunca bana her türlü imkânı sunan, her aşamada beni yönlendiren değerli danışmanım Sayın Prof. Dr. Selma ÜLGENTÜRK’e ve eş danışmanım Sayın Prof. Dr. Şerife BAYRAM’a teşekkürlerimi sunarım. Tez izleme komitesinde görev alan ve çalışmamın her aşamasında yardımlarını esirgemeyen, beni yönlendirerek çalışmalarıma katkı sağlayan kıymetli hocam sayın Prof. Dr. Sultan ÇOBANOĞLU’na, tez konusunun belirlenmesi de dahil olmak üzere çalışmanın her adımında var olan ve değerli katkılarını sunarak, bütün sorularıma büyük bir sabır ve nezaketle cevaplar verirken kendi cevaplarımı bulmam için de yol gösteren kıymetli hocam sayın Prof. Dr. İ. Alper SUSURLUK’a teşekkürlerimi sunarım. Tez savunma jürimde yer alarak değerli öneri ve katkıları ile tezimin zenginleşmesini sağlayan sayın hocam Prof. Dr. Ramazan CANHİLAL’e teşekkürlerimi sunarım. Hem çalışmalarımı gerçekleştirmem için Moleküler Entomoloji Laboratuvarı (MOLEN)’ndeki her türlü imkandan faydalanmamı sağlayan hem de tez savunma jürim olarak değerli görüş, öneri ve katkılarını paylaşan sayın hocam Doç. Dr. Umut TOPRAK’a teşekkürü bir borç bilirim. Çalışmalarımın her aşamasında katkı, öneri ve yardımlarını esirgemeyen, kendimi çıkmazda hissettiğim her durumda moral ve motivasyonumu yükselten değerli arkadaşım Arş. Gör. Emre İNAK’a ve her konuda fikir alışverişi yapabildiğim ve koşulsuz desteğini aldığım değerli arkadaşım Arş. Gör. Ebubekir YÜKSEL’e çalışmalarımın finansmanını sağlayan Ankara Üniversitesi Bilimsel Araştırma Projeleri Koordinatörlüğü’ne teşekkürlerimi sunarım. Doktora çalışmalarım ve hayatım boyunca bana gösterdikleri sevgi, saygı, anlayış, sabır ve destek için sevgili eşim Dr. Hasan Fehmi ÖZDEMİR’e, gözbebeğim DAĞLAR’a ve sevgili aileme sonsuz teşekkür ederim. iv İçindekiler ÖZET ........................................................................................................................................... ii ABSTRACT ................................................................................................................................ iii TEŞEKKÜR ............................................................................................................................... iv ÇİZELGELER DİZİNİ ............................................................................................................ vii ŞEKİLLER DİZİNİ ................................................................................................................. xiii SİMGELER DİZİNİ ............................................................................................................... xvii 1. GİRİŞ ...................................................................................................................................... 1 2. KURAMSAL TEMELLER .................................................................................................. 6 2.1 Nematodların Genel Özellikleri ........................................................................................... 6 2.2 Entomopatojen Nematodlar ................................................................................................. 9 2.2.1 Taksonomileri ..................................................................................................................... 9 2.3 Entomopatojen Nematodların Hayat Döngüleri .............................................................. 10 2.4 Bakteri- Nematod Simbiyotik İlişkisi ................................................................................ 12 2.5 Entomopatojen Nematodların Konukçuları ile Etkileşimleri ......................................... 17 2.6 Entomopatojen Nematodların Dağılımları ve Toprak İçerisindeki Konumları ........... 18 2.7 Entomopatojen Nematodlar ve Simbiyotik Bakterilerin Tarihçesi ve Yapılan Çalışmalara Genel Bir Bakış ............................................................................................. 20 3. MATERYAL VE YÖNTEM ............................................................................................... 39 3.1 Galleria mellonella Larvalarının Üretilmesi ..................................................................... 39 3.2 Toprak Örneklerinin Alınması .......................................................................................... 40 3.3 Toprak Örneklerinden Entomopatojen Nematod İzolasyonu ........................................ 44 3.4 Entomopatojen Nematodların Tür Teşhislerinin Yapılması .......................................... 46 3.4.1 Nematodların daimi preparatlarının yapılması ............................................................ 46 3.4.2 Morfometrik ölçümler ...................................................................................................
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