Forty-six of the entomopathogenic fungus Verticillium lecanii form various worldwide locations, from insect hosts, phytopathogen, plant and soil were investigated their spore size and sporulation. Average width ranged from 3.0 to 4.0μm and showed continuous variation. The average length ranged from 5.8 to 10.5μm among the isolates. The average length of Vertalec and Mycotal, which were already commercialized for aphids and whitefly, were 7.9μm and 6.2μm respectively. Further, the mean conidia yield for all isolates ranged from 0.3 to 10.9 ×10⁷ conidia/cm². The mean conidia yields of Vertalec and Mycotal were 1.0 ×10⁷ conidia/cm² and 5.3 ×10⁷ conidia/cm², respectively. All isolates were bioassayed against the green peach aphids (Myzus persicae) in the laboratory. Virulence toward the aphids of V. lecanii ranged from 15.2-96.5% apterous mortality 4 days after treatment with conidial suspensions. There was no relationship between geographical location and virulence. However, several isolates from whitefly and Mycotal had lower virulence, causing 15.2-48.9% mortality. Furthermore, five isolates have larger conidia dimensions than Vertalec and yielded lower numbers of conidia than Vertalec. Theses five isolates gave a higher mortality rate of aphids. There was a positive correlation among conidial morphology, sporulation and virulence toward aphids.
　In order to clarify relationships among genetic diversity, virulence, and other characteristics of conidia, forty-six isolates of Verticillium lecanii from various hosts and geographical locations were examined. DNA polymorphisms in ribosomal DNA (rDNA) of those isolates were analyzed using polymerase chain reaction (PCR). The internal transcribed spacer (ITS) and intergenic spacer (IGS) regions of rDNA were analyzed by PCR-RFLP (restriction fragment length polymorphism). The size of the PCR product from the ITS region was 580bp in 39 of the isolates. A 600bp ITS product was detected in 6 isolates. One Japanese isolate produced both the 580bp and 600bp products. Enzymatic digestion of the ITS region with Sau3AI, MspI, HaeIII and RsaI revealed RFLPs that consisted of 15 haplotypes. Four PCR amplification products were detected in the IGS region, with sizes ranging from ca 1.9 to 4.0kb. 18 distinct IGS haplotypes were detected in the IGS region. There were no correlations among the ITS or IGS haplotypes, isolation source, and location. When results from ITS and IGS haplotypes are combined, the 46 V. lecanii isolates except three were classified into 30 haplotypes. This result suggests that the haplotype of rDNA-RFLP analysis can be used for studying genetic diversity in V. lecanii. Furthermore, amplified product size of IGS (4.0-1.9 kb) did show relation with conidia size and virulence. Six isolates with 4.0-kb IGS products had large conidia dimensions, yielded low numbers of conidia compared with other isolates, and tended to have higher virulence against green peach aphids. These data demonstrate the level of genetic diversity of V. lecanii, and suggest relations among the genetic properties, conidial morphology, and virulence.
　In order to clarify relationships among genetic diversity, virulence, and other characteristics of conidia, forty-six isolates of Verticillium lecanii from various hosts and geographical locations were examined. The mitochondrial small subunit rDNA (mt-SrDNA) and β-tubulin were analyzed by PCR-RFLP. PCR-single stranded conformational polymorphism (SSCP) was also performed on regions of the mitochondrial large subunit rDNA (mt-LrDNA), mt-SrDNA, β-tubulin and histone 4 (H4). There were no relationships among the results of RFLP, SSCP, isolation source, and location. However, the SSCP patterns of mt-LrDNA and SSCP and RFLP patterns of β-tubulin divided isolates into the same groups as did IGS typing, separating type S from types M and L. These molecular observations were closely related with conidial morphological characteristics and virulence toward aphids. The one isolates with a 1.9 kb IGS product also had a unique haplotype in theses same analyses. Furthermore, double-stranded RNA (dsRNA) was detected in 22 out of 35 V. lecanii isolates and correlated with the amplicon sizes of IGS, though not with virulence or isolation location. Isolates containing dsRNA were divided into six distinct types based on banding pattern. These data demonstrate the level of genetic diversity of V. lecanii, and suggest relations among the genetic properties, conidial morphology, and virulence. Forty-three isolates of Verticillium lecanii from insects, phytopathogenic fungi and other substrates were tested for vegetative compatibility by observing heterokaryon formation among complementary nitrate-nonutilizing (nit) mutants. Only one isolate was unable to yield nit mutants. Among the others of V. lecanii, 21 isolates were self-incompatible. The remaining 21 self-compatible isolates were divided into 14 vegetative compatibility groups (VCGs): 10 VCGs were single-member VCGs; the remaining 4 VCGs (VL-J1, -J2, -J3 and -J9) included two to four isolates each. The results of present and previous chapter indicated that these four VCGs showed the same IGS haplotype. Further, these VCGs were correlated with a part of fragment pattern of other region (mt-LrDNA, -SrDNA, Bt-2 and H4 region) by PCR-RFLP and -SSCP, but not dsRNA detection. Two isolates belonging to VL-J2 showed high virulence to aphids, but this was not true for VL-J1 isolates. These findings indicate that two VCGs (VL-J1 and J2) may originate from two distinct clonal lineages. Alternatively, high VCG diversity and HSI frequency of V. lecanii may be associated with an array of distinct lineages. These data not only suggest relation between DNA polymorphisms, virulence, and VCG, but also demonstrate genetic heterogeneity of V. lecanii.

（和文要旨）
　糸状菌Verticillium lecaniiは昆虫・植物病原菌に寄生性を示し、生物防除剤として利用されている。本菌の宿主範囲は 広範囲であるが、各宿主に対して個々の菌株が、どの様なメカニズムで寄生性の程度を決定しているかは不明である。既に 本菌の生物防除剤として商品化されているアブラムシ用Vertalecとコナジラミ用Mycotalは、それぞれ大型・小型の分生子 系統で、胞子形成量やアブラムシとオンシツコナジラミに対する寄生性程度が異なっている(Hall, 1984)。本研究では、 大型・小型分生子系統の寄生性の違いにおけるメカニズムを解明する為にも、本菌のDNAレベルでの違いを調査し、胞子 特性との関連性を検討することを目的とした。

1. 本研究では、様々な宿主から分離された国内および外国産分離株、計46菌株を用いて、分生子の形態・形成量について 調査した。その結果、胞子長径と形成量間には-0.53の負の相関関係が示された。また、従来大型分生子系統とされていた Vertalecよりも胞子長径が長く、かつ胞子形成量が低い5菌株が存在した。次に、各供試菌株のモモアカアブラムシに 対する致死率を調査したところ、アブラムシ分離菌株に比べて、オンシツコナジラミ分離菌株とMycotalは低い致死率を 示した。Vertalecよりも胞子長径が長い菌株の内3菌株は、90%以上の高い致死率を示した。

2. 分生子や寄生性の特徴とDNAレベルでの関連性を検討するために、供試菌株のDNA多型を解析した。まず、核の リボゾームRNA遺伝子(rDNA)のスペーサー領域であるITS・IGS領域をPCR法により増幅した。その結果、ITS領域では、 1菌株を除く全ての国内産菌株から、580bpの産物が増幅されたのに対し、外国産菌株は580bpと600bpの2パターンが増幅された。 一方、IGS領域の増幅産物は4タイプ(1.9, 2.3, 3.0および4.0kb)得られた。増幅産物4.0kb(IGS-L型)を示す6菌株の中には、 Vertalecよりも胞子長径が長く形成量の少ない菌株が存在し、胞子特性と関連性が認められた。これら両領域の増幅産物を 各々RFLP解析したところ、バンドパターンに違いが認められ、その組み合わせによるrDNAハプロタイプのグループ分けは 個体群識別に利用できることが明らかになった。

3. 非転写領域だけでなくミトコンドリアDNA内のrDNAラージサブユニット(mt-LrDNA)、スモールサブユニット、核の β-チューブリン遺伝子(Bt-2)、ヒストン4遺伝子のコード領域についてPCR-RFLPおよび-SSCP解析を実施し、胞子特性や その他の形質との関連性を調査した。その結果、mt-LrDNAのSSCP解析、Bt-2のRFLP、SSCP解析によるバンドパターンの違いが、 IGS増幅産物の4タイプと一致し、かつ胞子特性との関連性が認められた。dsRNAは分生子による垂直伝搬か菌糸融合による 水平伝搬のみで移行することから、菌株間の関連性を調査するためdsRNAの検出を試みたところ、35供試菌株中22菌株から dsRNAが検出された。dsRNAのフラグメントパターンは菌株間で異なっており、IGS-L型の3菌株は他の菌株とは異なる特異的な パターンを検出した。

4. 本菌を始め不完全菌類の分類において遺伝的類縁関係を調査する方法として栄養体親和性試験が用いられている。 そこでKClO₃含有培地を用いて、nit (硝酸還元能欠損変異株を分離した。栄養体親和性グループ(VCG)の判定はnit1と NitMのヘテロカリオン親和性の程度で判断した。その結果、21菌株が自己不親和性を示し、残り21菌株は自己親和性を 示した。この21自己親和性株は、14VCGsを構成し、この内10VCGsは、各々1菌株のみを含み、残り4VCGsについては2-4菌株を 含んでいた。これら4VCGs(VL-J1, -J2, -J3, -J9)のIGSハプロタイプはグループ内で各々一致していた。また、VL-J1に 属するオンシツコナジラミ分離株は、モモアカアブラムシに対する致死率が低かったのに対し、VL-J2に属する ダイコンアブラムシ分離株は、致死率が高い傾向にあった。

　以上のことから、本菌の分生子形態と胞子形成量間に相関関係が認められ、更に、この胞子特性はIGS領域の増幅産物の 違いや他の領域におけるDNA多型およびアブラムシに対する寄生性との関連性があることが明らかになった。これらの関連性は、 寄生性分化のメカニズムを解明するうえで重要である