香榧绿藻的生物学特性及物种鉴定

香榧绿藻Chlorella sp.是一种附着在香榧Torreya grandis ‘Merrillii’枝、叶上的藻类。为探明香榧绿藻的种属类别及亲缘关系，探究香榧绿藻的生物学特性，以3个不同地区的香榧树上采集的香榧绿藻为试验对象，通过室内培养，观察香榧绿藻的形态特征及生活习性，提取香榧绿藻DNA并进行18S rDNA序列和内转录间隔区（ITS）基因序列特异性扩增，最终序列进行BLAST比对，绘制系统进化树。结果表明:香榧绿藻由A组和B组2种不同形态的绿藻组成，不同地区香榧绿藻形态存在差异，但主要以A组形态绿藻为主。根据形态学鉴定结果表明:A组香榧绿藻与引起番茄绿藻病的椭圆小球藻Chlorella ellipsoidea形态相近，B组香榧绿藻与引起黄瓜绿藻病的丝藻Ulothrix sp.形态相近；但香榧绿藻对pH值的反应规律与斜生栅藻Scenedesmus obliquus相近。通过序列比对发现，2种形态的香榧绿藻的18S rDNA和ITS区基因序列也与栅藻科Scenedesmace的Asterarcys quadricellulare的同源性高，相似率达100%。结合传统形态学方法和分子生物学技术，将A组香榧绿藻初步鉴定为栅藻科的Asterarcys quadricellulare，B组香榧绿藻为其变种，且2组香榧绿藻与同属于栅藻科的Scenedesmus sp. SM15_4（KT778094.1）的ITS区基因序列相同。香榧绿藻具有较好的耐碱性，生长周期长且不容易腐烂。这些生物学特性赐予香榧绿藻在香榧树上长期寄生的能力。

Biological characteristics and species identification of Chlorella sp. with Torreya grandis 'Merrillii'

Chlorella sp. is an algal attached to the branches and leaves of Torreya grandis 'Merrillii'. To explore the relationship the Chlorella sp. to Torreya grandis 'Merrillii' and to study the biological characteristics of the Chlorella sp., the Chlorella sp. was collected from T. grandis 'Merrillii' trees in three different regions and tested. According to an indoor culture, the morphological characteristics and living habits of the Chlorella sp. were observed, and the DNA of the Chlorella sp. extracted had a sequence specific amplification of 18S rDNA. An Internal Transcribed Spacer (ITS) gene sequence was also performed. The final sequence was aligned with the Basic Local Alignment Search Tool (BLAST) to draw a phylogenetic tree in neighbor-joining (NJ) method with three replications. Results showed two different forms of green algae, A and B. There were differences in the species of green algae in different areas, but they were mainly in the form of the A-type green algae. According to morphological identification results, the A-type Chlorella sp. was similar to the morphology of Chlorella ellipsoidea which could cause tomato green algae disease; the B-type Chlorella sp. was similar to the morphology of Ulothrix sp. which could cause cucumber green algae disease. However, the response of Chlorella sp. to pH was similar to that of Scenedesmus obliquus. The sequence alignment showed that the 18S rDNA and ITS gene sequences of the two forms of Chlorella sp. also had a high homology with Asterarcys quadricellulare of the Scenedesmaceae family with the similarity rate being 100%. Combining traditional morphological methods and molecular biology techniques, group A Chlorella sp. was identified as Asterarcys quadricellulare of the Scenedesmaceae family and group B Chlorella sp. identified as its variant. The two groups of Chlorella sp. had the same ITS region gene sequence as Scenedesmus sp. SM15_4 (KT778094.1) belonging to the Scenedesmaceae family. In conclusion, Chlorella sp. had favorable biological properties such as alkali resistance, a long growth cycle, and resistance to decay which helped it live on the Torreya grandis 'Merrillii' tree for a long time.