Root Rot And Its Biological Control Of Lucerne Growing At Huanxian County, Gansu Province

Lucerne(Medicago sativa) is a highly nutritious animal feed and globally is one of most important forage crops. The plant plays an important role in crop rotation and is a good green manure, which can control water and soil erosion as well as having positive effects on soil fertility,soil structure, and soil health. The root rot is one of limiting factors to lucerne production throughout the world and is difficult to control. In this study root rot disease was investigated in the lucerne(Medicago sativa cv. Longdong) fields of Huanxian county, Gansu Province. The fungi associated with root rot in the lucerne samples collected were isolated and examined by morphological characteristics and molecular assays. PCR assays were established for the causal agents. In order to control the disease experiment was set out to discover antagonistic microorganisms for use as a potential biocontrol agent. The main results are as follows:1. Fifteen root-invading fungi were isolated from the roots showing root rot symptoms and identified through microscopy, culture methods, and utilizing PCR and DNA sequencing techniques. The fungal species, in order of decreasing frequency of isolation, were Fusarium semitectum, Phoma medicaginis, F. equiseti, Alternaria alternata, F. acuminatum, Scopulariopsis sp., Penicillium sp., Microdochium tabacinum, Collectotrichum destructivum, Chaetomium sp., F.oxysporum, Podospora sp., Chaetosphaeronema sp., F. avenaceum, and A. tenuissima. Among the15 fungal species, Fusarium spp. was considered the main pathogen with the highest isolation rate of 51.9%. This is the first report of M. tabacinum and Chaetosphaeronema sp. being pathogens of lucerne in China.2. The pathogenicity of eleven fungal isolates including A. alternata, A. tenuissima,Chaetosphaeronema sp., C. destructivum, F. acuminatum, F. equiseti, F. oxysporum, F.semitectum, M. tabacinum, P. medicaginis, and Podospora sp. isolated from lucerne roots was tested in greenhouse by soil inoculation method. F. oxysporum, followed by F. semitectum,resulted in the largest disease index on lucerne. A. alternata, A. tenuissima, Chaetosphaeronema sp., C. destructivum, F. acuminatum, F. equiseti, M. tabacinum, and P. medicaginis were also virulent in various extend, and Podospora sp. was non-pathogenic to lucerne. The 10 pathogens induced typical symptoms of lower leaves wilting, brown lesions on roots and root rot on lucerne,with disease index ranged 4.4~65.4. Compared with the uninoculated control, the dry weights of shoot and root of diseased lucerne 30 days after inoculation were decreased by 8.4%~60.3% and16.0%~59.6%, respectively, the length of shoot and root was decreased by 13.1%~52.4% and3.6%~49.6%, respectively. F. semitectum, followed by C. destructivum, resulted in the largest disease index on white clover(Trifolium repens). The results indicated these inoculated fungi were all induced root lesions on white clover, with disease index 3.2~32.3. The dry weights of shoot and root of deseased white clover 30 days after inoculation were decreased by 8.1%~65.0% and7.1%~51.8%, respectively. The greenhouse experiments also showed that the pathogenic effects on plants tested decreased as the plant growth 60 days after inoculation and afterwards.3. Host range of M. tabacinum and C. destructivum were determined using culture plate method. The result showed that M. tabacinum was pathogenic to lucerne, cucumber(Cucumis sativus), zuchini(Cucurbita pepo), pumpkin(Cucurbita moschata), melon(Cucumis melo), and water melon(Citrullus lanatus), the disease incidence and disease index was 70.5%~100% and33.1 ~ 91.8, respectively. C. destructivum was pathogenic to lucerne, sweet pepper(Capsicum annuum), tomato(Lycopersicon esculentum), radish(Raphanus sativus), cabbage(Brassica oleracea), pea(Pisum sativum), and bean(Phaseolus vulgaris), with 33.3% ~ 100% disease incidence and 8.3~50.4 disease index, respectively.4. A polymerase chain reaction(PCR) assay based on the ITS(Internal transcribed spacer)region of the rDNA was developed to amplify a 304 bp fragment from DNA concentrations as low as 20 fg/μl, which was sensitive enough to detect M. tabacinum in diseased root samples.A Real-time PCR assay based on the TEF-1а gene and SYBR Green I technology was established for the causal agents F. semitectum and F. acuminatum. Pure cultures of F. semitectum and F. acuminatum were detectable in the Real-time PCR assay. The assay was also successful in detecting F. semitectum and F. acuminatum in lucerne roots in growth-chamber experiments. The SYBR Green I-based Real-time assay was able to quantify F. semitectum and F. acuminatum down to 35.6 fg/μL and 87 fg/μL, respectively.5. A bacterial strain MB29 obtained from lucerne roots was identified as Bacillus subtilis subsp. spizizenii using Biolog Microlog microbial identification system and sequence analysis of16 S rDNA gene. The ability of strain MB29 to inhibit 10 root-invading fungi isolated from lucerne roots in previous experiments was examined using culture plate tests. Mycelium growth of the test fungi(except Chaetomium sp.), including A. alternata, A. tenuissima, C. destructivum, F.acuminatum, F. equiseti, F. oxysporum, F. semitectum, M. tabacinum, and P. medicaginis, was reduced by 33.4%~ 90.2%. The maximum inhibitory effect of MB29 was observed against A.alternata, followed by C. destructivum and F. semitectum, with the mycelium growth beingreduced by 90.2%, 85.5% and 84.5%, respectively. The severity of F. semitectum on lucerne seedlings treated with MB29 was decreased by 43.4% and seedling fresh weight increased by37.1%, compared with the nontreated control.