|
|
Journal of Zhejiang University SCIENCE B
ISSN 1673-1581(Print), 1862-1783(Online), Monthly
2014 Vol.15 No.11 P.966-978
Designing a SCAR molecular marker for monitoring Trichoderma cf. harzianum in experimental communities
Abstract: Several species of the fungal genus Trichoderma establish biological interactions with various micro- and macro-organisms. Some of these interactions are relevant in ecological terms and in biotechnological applications, such as biocontrol, where Trichoderma could be considered as an invasive species that colonizes a recipient community. The success of this invasion depends on multiple factors, which can be assayed using experimental communities as study models. Therefore, the aim of this work is to develop a species-specific sequence-characterized amplified region (SCAR) marker to monitor the colonization and growth of T. cf. harzianum when it invades experimental communities. For this study, 16 randomly amplified polymorphic DNA (RAPD) primers of 10-mer were used to generate polymorphic patterns, one of which generated a band present only in strains of T. cf. harzianum. This band was cloned, sequenced, and five primers of 2023 mer were designed. Primer pairs 2F2/2R2 and 2F2/2R3 successfully and specifically amplified fragments of 278 and 448 bp from the T. cf. harzianum BpT10a strain DNA, respectively. Both primer pairs were also tested against the DNA from 14 strains of T. cf. harzianum and several strains of different fungal genera as specificity controls. Only the DNA from the strains of T. cf. harzianum was successfully amplified. Moreover, primer pair 2F2/2R2 was assessed by quantitative real-time polymerase chain reaction (PCR) using fungal DNA mixtures and DNA extracted from fungal experimental communities as templates. T. cf. harzianum was detectable even when as few as 100 copies of the SCAR marker were available or even when its population represented only 0.1% of the whole community.
Key words: Trichoderma cf. harzianum, Sequence-characterized amplified region (SCAR), Molecular marker, Experimental fungal communities
The online version of this article contains supplementary materials Figs. S1-S5, FASTAs S1-S2, Table S1
创新要点：多种木霉属真菌能与各种微观和宏观的生物有机体建立相互作用。利用这些相互作用，木霉可做为原生种群的入侵物种而用于生物防治。本文通过使用试验菌群为研究模型，利用随机扩增多态性DNA（RAPD）技术和序列特征性扩增区域（SCAR）标记物来监测菌群中哈茨木霉的生长状态。
研究方法：利用随机扩增多态性DNA（RAPD）技术，从16个10聚体引物进行多态性筛选，其中1个引物扩增出对应哈茨木霉的条带。对该条带进行克隆测序，并设计5个20-23聚体引物。成功利用引物对2F2/2R2和2F2/2R3278分别特异性地扩增出哈茨木霉BpT10a菌株278 bp和448 bp的DNA片段。同时，用这两个引物对14个哈茨木霉菌株和几种不同的真菌菌株进行特异性对照试验，也只成功扩增出哈茨木霉菌株。此外，使用真菌DNA混合物和试验真菌群的DNA为模板，采用实时聚合酶链式反应（PCR）对引物对2F2/2R2进行评估。当仅使用100份SCAR标记物或哈茨木霉仅占整个菌群的0.1%时，仍能检测出哈茨木霉。
重要结论：本研究所建立的SCAR分子标记能有效监测菌群中的哈茨木霉的定殖和生长，具有较高特异性、灵敏度和准确度。
关键词组：
References:
Open peer comments: Debate/Discuss/Question/Opinion
<1>
DOI:
10.1631/jzus.B1400063
CLC number:
Q93; Q78
Download Full Text:
Downloaded:
2652
Download summary:
<Click Here>Downloaded:
1804Clicked:
6984
Cited:
2
On-line Access:
2014-11-04
Received:
2014-03-03
Revision Accepted:
2014-07-22
Crosschecked:
2014-08-25