Identification of the crayfish plague fungus Aphanomyces astaci by polymerase chain reaction and restriction enzyme analysis

To characterise the DNA of the crayfish plague fungus Aphanomyces astaci, Saprolegniales (Oomycetes), primers were developed to amplify a 1050bp segment of the 28S rDNA region. Restriction enzymes were applied to the amplicon obtained, to distinguish A. astaci from 12 fungal species belonging also to the Saprolegniales and five more distantly related fungi. Most of the fungal species included in the study are either known parasites of freshwater crayfish cuticle or can be found in their natural environment. A. astaci DNA was distinguishable from the DNA of other fungal species tested by using the primers developed plus restriction enzymes AluI, HindIII and AvaI.Prior to this study, methods for A. astaci-species determination, e.g. spore production and infection experiments, required a protracted period to yield results; the method described in this study is quicker.     

Establishment of TaqMan Real-time PCR Assay for Detection of Arcanobacterium pyogenes

The study was aimed to establish a specific and sensitive TaqMan Real-time PCR assay for detection of Arcanobacterium pyogenes (A.pyogenes).Based on the conservative sequence of pyolysin (PLO) gene of A.pyogenes published in GenBank, specific primers and TaqMan probes were designed. The TaqMan Real-time PCR assay was established, and the specificity and sensitivity were tested.The specificity test results showed that only A.pyogenes exhibited typical curves.The detection sensitivity of this assay was 77.6 fg genomic DNA per 20 μL reaction, and 63 CFU/mL for pure cultures.16 out of 23 clinical samples were positive detected by the TaqMan Real-time PCR assay, which were consistent with API Coryne identification.The TaqMan Real-time PCR assay developed in this study was specific and sensitive for detection of A.pyogenes, and it could be used for identification and epidemiological investigation of A.pyogenes.     

化脓隐秘杆菌TaqMan实时荧光定量PCR检测方法的建立

本试验旨在建立检测化脓隐秘杆菌(Arcanobacterium pyogenes,A.pyogenes)特异、灵敏的TaqMan实时荧光定量PCR检测方法。根据GenBank公布的化脓隐秘杆菌溶血素(pyolysin,PLO)基因高保守序列,设计特异性引物和探针建立检测体系,用于化脓隐秘杆菌的快速检测,并对该方法的特异性和灵敏度进行检测。结果显示,本试验建立的TaqMan实时荧光定量PCR方法仅对化脓隐秘杆菌的检测结果为阳性;该方法最低检测DNA浓度为77.6 fg,最低检测细菌浓度为63 CFU/mL。采用本研究建立的方法检测23份林麝临床病例样品,共鉴定出16株化脓隐秘杆菌,与API Coryne生化鉴定方法的结果相同。本研究为化脓隐秘杆菌的检测提供了一种灵敏、特异、快速的检测方法,其可用于化脓隐秘杆菌的诊断和流行病学调查。     

山羊草属杀配子染色体2C特异SCAR标记的建立

用40条10碱基随机引物,对普通小麦“中国春”、“中国春”-杀配子染色体2C二体附加系、柱穗山羊草进行RAPD分析,筛选到1个杀配子染色体2C特异引物OPF03。从“中国春”-杀配子染色体2C二体附加系中克隆了该DNA片段。测序结果表明,该片段长1 496 bp,与大麦1个cDNA的同源性为92%。根据OPF03₁₄₉₆的序列设计了1对SCAR引物2C-F₅₈₆、2C-R₅₈₆,对普通小麦“中国春”、“中国春”-杀配子染色体2C二体附加系、柱穗山羊草、二倍体长穗偃麦草、“中国春”-长穗偃麦草7E二体附加系等材料进行了SCAR分析,在“中国春”-杀配子染色体2C二体附加系、柱穗山羊草中扩增出了586 bp的片段,而在普通小麦“中国春”、二倍体长穗偃麦草、“中国春”-长穗偃麦草7E二体附加系中没有该产物,初步证明此标记可以用于杀配子染色体2C的检测。进一步用该对SCAR引物对“中国春”-杀配子染色体2C二体附加系与“中国春”-长穗偃麦草7E二体附加系的杂种F₁代、F₂代进行了分析,结果在全部F₁代以及部分F₂代材料扩增出了目的片段,进一步证明了此SCAR标记可以用来检测小麦背景下的杀配子染色体2C,为小麦背景中杀配子染色体2C的快速跟踪检测提供了新途径。     

Real-time PCR as a surveillance tool for the detection of Trichinella infection in muscle samples from wildlife

Trichinella nematodes are the causative agent of trichinellosis, a meat-borne zoonosis acquired by consuming undercooked, infected meat. Although most human infections are sourced from the domestic environment, the majority of Trichinella parasites circulate in the natural environment in carnivorous and scavenging wildlife. Surveillance using reliable and accurate diagnostic tools to detect Trichinella parasites in wildlife hosts is necessary to evaluate the prevalence and risk of transmission from wildlife to humans. Real-time PCR assays have previously been developed for the detection of European Trichinella species in commercial pork and wild fox muscle samples. We have expanded on the use of real-time PCR in Trichinella detection by developing an improved extraction method and SYBR green assay that detects all known Trichinella species in muscle samples from a greater variety of wildlife. We simulated low-level Trichinella infections in wild pig, fox, saltwater crocodile, wild cat and a native Australian marsupial using Trichinella pseudospiralis or Trichinella papuae ethanol-fixed larvae. Trichinella-specific primers targeted a conserved region of the small subunit of the ribosomal RNA and were tested for specificity against host and other parasite genomic DNAs. The analytical sensitivity of the assay was at least 100 fg using pure genomic T. pseudospiralis DNA serially diluted in water. The diagnostic sensitivity of the assay was evaluated by spiking 10 g of each host muscle with T. pseudospiralis or T. papuae larvae at representative infections of 1.0, 0.5 and 0.1 larvae per gram, and shown to detect larvae at the lowest infection rate. A field sample evaluation on naturally infected muscle samples of wild pigs and Tasmanian devils showed complete agreement with the EU reference artificial digestion method (k-value=1.00). Positive amplification of mouse tissue experimentally infected with T. spiralis indicated the assay could also be used on encapsulated species in situ. This real-time PCR assay offers an alternative highly specific and sensitive diagnostic method for use in Trichinella wildlife surveillance and could be adapted to wildlife hosts of any region.     

冰草组织培养再生体系建立及耐旱转基因研究

本研究以冰草属植物中的4个品种——蒙古冰草新品系(A.mongolicum Keng)、航道冰草(A.cristatum cv.Fairway)、诺丹冰草(A.desertorum cv.Nordan)和一个种间杂种冰草——蒙农杂种冰草(A.cristatum×A.desertorum cv.Hycrest-Mengnong)为材料,分别以幼穗为外植体建立了冰草组织培养再生体系。在此基础上,以调控脯氨酸生物合成最后一步的关键酶的突变体基因P5CS为目标基因,bar基因为筛选标记基因,用基因枪法轰击幼穗诱导的愈伤组织,获得转基因植株。利用PCR方法从拟南芥(Arabidopsis thaliana)基因组DNA中分离得到了CBF4及其启动子,并对CBF4启动子进行了序列分析和初步的功能分析。     

Evaluation of conventional and real-time PCR assays for detection and differentiation of Spotted Fever Group Rickettsia in dog blood

Spotted Fever Group Rickettsia is important cause of emerging and re-emerging infectious disease in people and dogs. Importantly, dogs can serve as sentinels for disease in people. Sensitive and specific diagnostic tests that differentiate among species of infecting Rickettsia are needed. The objective of this study was to develop a sensitive and specific PCR that differentiates SFG Rickettsia infecting dog blood. Conventional and real-time PCR assays were developed using primers that targeted a small region of the ompA gene. Their sensitivity, determined by testing a cloned target sequence in the presence of host DNA, was 15–30 and 5 copies of DNA, respectively. Testing of Rickettsia cultures and analysis of Rickettsia gene sequences deposited in GenBank verified DNA could be amplified and used to differentiate species. DNA from the blood of infected dogs was also tested. Importantly, Rickettsia DNA was detected before seroconversion in some dogs. The species of infecting Rickettsia was also identified. We conclude these assays may assist in the timely diagnosis of infection with SFG Rickettsia. They may also facilitate the discovery of novel SFG Rickettsia infecting dogs, and in the investigation of dogs as sentinels for emerging rickettsioses.     

Anaplasma infection in free-ranging Iberian red deer in the region of Castilla-La Mancha, Spain

Organisms in the genus Anaplasma are obligate intracellular pathogens that multiply in both vertebrate and invertebrate hosts. The type species, Anaplasma marginale, causes bovine anaplasmosis and infects erythrocytes of the vertebrate host and undergoes a complex developmental cycle in ticks which serve as biological vectors. Infected cattle, wild ruminants and ticks can all serve as reservoirs of A. marginale. In this study, hunter killed Iberian red deer (Cervus elaphus hispanicus) from the region of Castilla-La Mancha in southwestern Spain were tested for Anaplasma infection. We found that 10% of the deer examined were seropositive for Anaplasma. Three A. marginale strains were subsequently obtained from salivary glands of Hyalomma marginatum that were removed from these deer, and the sequence of the major surface protein (msp)4 gene was determined for each strain and used for phylogenetic studies. Maximum parsimony analyses of msp4 sequences from H. marginatum ticks in comparison with New World cattle and bison isolates reported previously, suggested different origins for these Spanish A. marginale strains. The results of this study demonstrated that Iberian red deer are naturally infected with Anaplasma, and may therefore serve as a wildlife reservoir of the pathogen. Although the link between deer infection and the strains of A. marginale identified in ticks was not established, H. marginatum and Rhipicephalus bursa were identified as potential biological vectors for A. marginale in this region and may effect transmission of A. marginale between deer and cattle populations.     

Differential diagnosis of dog hookworms based on PCR-RFLP from the ITS region of their rDNA

Species of Ancylostoma infecting dogs and sometimes humans are sympatric in many parts of the world. The establishment of a specific molecular diagnostic tool is important, not only to refine information for epidemiological studies, but also to evaluate the efficacy of vaccine programmes and assist in the development of specific drug treatments. The ITS region from 20 specimens of A. braziliense, collected from three separate geographical areas of Brazil, and from 10 specimens of A. caninum, collected from the same area in Brazil were sequenced and analyzed. Alignment of sequences showed that this gene is highly conserved. The intraspecific polymorphism for both species was less then 1%, whereas the interspecific polymorphism was 6.2, 7.3 and 9.4% between A. ceylanicum and A. braziliense; A. caninum and A. ceylanicum and A. ceylanicum and A. braziliense, respectively. Among the three species it was 12.3%. This revealed the ITS region as highly conserved and consequently a good molecular marker for diagnostic studies. In this work, four restriction enzymes were used in a PCR-RFLP using the ITS region of rDNA, to establish a differential diagnosis which discriminates between three Ancylostoma species, A. braziliense, A. caninum and A. ceylanicum. The best pattern was given by the HinfI enzyme, which produced different fragment sizes for each of the three species. Furthermore, the diagnostic tool differentiates DNA extracted directly from faeces of Ancylostoma-infected dogs.     

Prevalence of bovine haemoparasites in Aceh province of Northern Sumatra: Implications for imported cattle

A limited seroepidemiological investigation was conducted in Aceh Province to determine the seroprevalence of Anaplasma marginale and Trypanosoma evansi in indigenous cattle and in groups of Sahiwal cross cattle imported from New Zealand in 1985. The results obtained suggest that these parasites are endemic in the areas surveyed; in local cattle, overall prevalence rates of 82% for A. marginale and 61% for T. evansi were obtained.

Thin blood smears were prepared from 42 anaemic adult cattle and 19 clinically normal calves. Babesia bigemina was detected in 5 blood smears and Theileria orientalis in 51. Sahiwal cattle imported from New Zealand suffered high mortalities during their first year in Aceh and it is suggested that imported naive cattle rapidly become infected with blood parasites.     

Development and evaluation of a quantitative PCR assay for detection of Hepatozoon sp

With the aim to improve current molecular diagnostic techniques of Hepatozoon sp. in carnivore mammals, we developed a quantitative PCR (qPCR) assay with SYBR Green I^®. The method, consisting of amplification of a 235 bp fragment of the 18S rRNA gene, is able to detect at least 0.1 fg of parasite DNA. Reproducible quantitative results were obtained over a range of 0.1 ng–0.1 fg of Hepatozoon sp. DNA. To assess the performance of the qPCR assay, DNA samples from dogs (140) and cats (50) were tested with either standard PCR or qPCR. Positive samples were always confirmed by partial sequencing of the 18S rRNA gene. Quantitative PCR was 15.8% more sensitive than standard PCR to detect H. canis in dogs. In cats, no infections were detected by standard PCR, compared to two positives by qPCR (which were infected by H. canis as shown by sequencing).     

二月兰种带真菌致病性研究

二月兰是重要的地被、景观和绿肥作物。通过种子、离体叶片和温室盆栽试验，对分离自二月兰种子的链格孢菌，芸薹生链格孢，细极链格孢，黑附球菌，变红镰刀菌，顶孢哈氏霉，细基格孢属等5属7种可培养真菌进行了致病性测定。结果表明，7种真菌可致二月兰种子萌发后烂种烂芽率16.50%~68.50%，其中芸薹生链格孢、顶孢哈氏霉可引致种子发芽率降低18.18%~27.27%。7种种带真菌分别引致二月兰离体叶片出现褪绿和坏死腐烂等症状，发病率100%，病斑面积8.84%~99.38%，病情指数22.50~95.00。盆栽条件下，种带真菌均可致植物出现萎蔫褪绿和坏死叶斑等症状，病株率100%，叶片发病率41.56%~79.88%，病情指数16.22~56.93。与对照（不接种带真菌）相比，种带真菌侵染二月兰植株后第9天植物丙二醛（MDA）含量增加30.40%~204.15%，过氧化物酶（POD）活性变幅为-1.81%~82.87%，超氧化物歧化酶（SOD）活性升高18.78%~86.14%，叶绿素含量（SPAD值）降低13.24%~37.85%。致病性试验表明，芸薹生链格孢致病性最强，顶孢哈氏霉致病性最弱。     

二月兰种带真菌分离与鉴定

种带真菌是植物病害的重要传播途径,本研究以重要绿肥作物二月兰（Orychophragmus violaceus）为研究对象,通过室内分离培养、形态学和分子学鉴定,旨在明确二月兰种带真菌种类,探讨其种带真菌与田间病害发生的相关性。试验结果获得二月兰种带真菌5属7种,分别为链格孢属链格孢菌（Alternaria alternata）,芸薹生链格孢（A. brassicicola）,细极链格孢（A. tenuissima）,附球菌属黑附球菌（Epicoccum nigrum）,镰刀菌属变红镰刀菌（Fusarium incarnatum）,哈氏霉属顶孢哈氏霉（Harzia acremonioides）,细基格孢属（Ulocladium sp.）真菌;其中芸薹生链格孢是导致田间二月兰叶斑病的病原。通过高通量测序得到二月兰种带真菌25属30种,包括常见致病菌芸苔链格孢、十字花科白粉菌（Erysiphe cruciferarum）、孔状短小茎点霉（Phoma exigua var. exigua）、粉红单端孢（Trichothecium roseum）和大丽轮枝菌（Verticillium dahliae）等。可见,二月兰种带真菌多为常见致病菌,应进一步开展其致病性研究,关注种带真菌与田间病害发生的相关性,据此研发防治技术。     

1种布鲁氏菌微滴式数字PCR检测方法的建立

本研究旨在构建1种布鲁氏菌（Brucella）微滴式数字PCR方法（droplet digital PCR，ddPCR）。以布鲁氏菌BCSP31基因为靶基因，选取保守区域设计引物和探针，构建了布鲁氏菌微滴式数字PCR方法。然后优化了ddPCR反应中的引物、探针浓度及退火温度，并进行方法的灵敏度、特异性和重复性试验。结果表明，ddPCR的最佳引物和探针浓度分别为900 nmol·L^-1和250 nmol·L^-1，最佳的退火温度为58℃。ddPCR的线性良好，最低检测下限为1.12 copies·μL^-1，与大肠杆菌O：157菌株等其他4种细菌无交叉反应，而且批内重复性和批间重复性都较好。综上表明，本研究建立的ddPCR方法灵敏度高、特异性强，可对布鲁氏菌感染的临床样品进行定量检测。     

羊源多杀性巴氏杆菌重组酶聚合酶扩增诊断方法的建立

为建立适用于羊源多杀性巴氏杆菌的重组酶聚合酶扩增（RPA）诊断方法,本研究依据前期测序鉴定的羊源多杀性巴氏杆菌KMT1基因序列构建pET-28a （＋）-KMT1质粒标准品。设计基于RPA技术的特异性引物及RPA荧光探针,建立实时荧光RPA方法的最适反应体系。采用10倍梯度连续稀释的质粒标准品检测该RPA方法的敏感性并绘制相关性曲线;以10种不同菌株的基因组DNA为模板验证方法的特异性;用感染多杀性巴氏杆菌的山羊及小鼠组织样品对方法的可靠性进行验证。结果显示,本试验建立的实时荧光RPA方法最适反应温度为39 ℃,最佳引物为KMT1-Fe1,灵敏度达100拷贝/μL,检测下限为10拷贝/μL。与大肠杆菌、金黄色葡萄球菌、副伤寒沙门氏菌、副溶血弧菌、绿脓杆菌、产酸克雷伯菌、布鲁氏菌S2株和鲍曼不动杆菌均无交叉反应。对13个组织样本进行检测,阳性率为76.9%,与实时荧光定量PCR检测结果的符合率达92.3%。综上所述,本研究建立的羊源多杀性巴氏杆菌实时荧光RPA方法具有特异性强、灵敏度高、可靠、快速便捷等特点,适用于多杀性巴氏杆菌的临床分子诊断。     

不同类型抗蒸腾剂对4种草本植物叶片水分利用效率的影响

为探究3种抗蒸腾剂叶片喷施对4种草本植物水分消耗和利用的影响规律,比较得出不同抗蒸腾剂的最佳使用浓度,为干旱荒漠区采煤迹地植被保育技术提供参考,本试验以蒙古冰草（Agropyron mongolicum Keng）、沙打旺（Astragalus adsurgens Pall.）、白花草木樨（Melilotus albus Medic.ex Desr.）、蜀葵（Althaea rosea （Linn.）Cavan.）4种草本植物为研究对象,每种草叶面分别喷施不同类型和浓度的抗蒸腾剂：旱地龙（Fulvic Acid,FA）浓度分别为1,2,3,4 g·L^-1;冠存（Guan Cun,GC）浓度分别为3,6,9,12 g·L^-1;高岭土（Kaolin,KL）浓度分别为20,40,60,80 g·L^-1,以喷施清水为对照,喷施3个月后测定其对4种草叶片蒸腾速率（Transpiration rate,Tr）、净光合速率（Net photosynthetic rate,Pn）、水分利用效率（Water use efficiency,WUE）和生物量的影响。结果表明：在试验浓度区间内,FA浓度越高,蒙古冰草、沙打旺、白花草木樨的Tr值越小、Pn值越小,WUE呈现先升高再降低的趋势;GC喷施浓度越高,蒙古冰草、沙打旺、白花草木樨叶片的Tr值越小、WUE值越大,Pn值较对照低但各浓度间未达到显著性差异;KL喷施浓度越高,蒙古冰草与蜀葵各浓度处理的Pn值越小、WUE值越小,Tr值较对照低但各浓度间未达到显著性差异。3种抗蒸腾剂对蒸腾速率的抑制程度均为GC和KL大于FA,对净光合速率的抑制程度均为KL和GC小于FA,对水分利用效率的提升程度均为KL和GC大于FA。综合3种抗蒸腾剂在植物叶面的喷施效果,GC和KL喷施效果较好,FA较差;蒙古冰草与沙打旺喷施12 g·L^-1的GC、白花草木樨与蜀葵喷施20 g·L^-1 KL的措施可用于干旱荒漠区采煤迹地的植被恢复。     

7种禾本科牧草抗旱性研究与评价

以7种多年生禾本科牧草（蒙古冰草-宁夏、蒙古冰草-内蒙古、沙生冰草、扁穗冰草、细茎冰草、格兰马草、老芒麦）为研究对象，系统分析了农艺性状（绿叶数和株高）收益与土壤水分收益的权衡关系，将权衡值（RMSD）与土壤含水量作为分位数模型变量，确定维持植物正常生长的土壤含水量的响应阈值，同时研究干旱胁迫下生理指标综合评价值，进行抗旱性评价。结果表明：1）干旱胁迫程度越高，牧草的绿叶数和株高受到的负面影响就越大；2）不同牧草对干旱胁迫的耐受程度和范围有差异，其中，细茎冰草和格兰马草的绿叶数-土壤含水量阈值分别为9.1%和9.7%，细茎冰草、格兰马草和蒙古冰草-宁夏的株高-土壤含水量阈值分别为4.6%、5.6%和13.0%，其余牧草分位数模型均未通过显著性检验；3）通过7项生理指标评价分析，7种多年生禾本科牧草抗旱性强弱表现为：蒙古冰草-宁夏>格兰马草>细茎冰草>蒙古冰草-内蒙古>沙生冰草>扁穗冰草>老芒麦；4） 综合土壤含水量阈值与抗旱生理评价，细茎冰草与格兰马草可作为中度干旱区的引选牧草。     

山黧豆根腐病病原菌亚洲镰孢菌的分离鉴定

为了明确山黧豆（Lathyrus sativus）根腐病病原菌的分类地位,本研究于2018年8月在四川省南充市采集山黧豆根腐病病样,经组织分离法进行分离纯化,采用形态学和翻译延伸因子（Translation elongation factor 1-agene,EF-1α）基因序列分析相结合的方法对致病菌进行鉴定。结果表明：从采集的山黧豆根腐病病样上共分离获得39株分离物,其中3株分离物Shan-27,Shan-28和Shan-29对山黧豆具有强致病性,分离频率为7.69%;这3株致病菌在形态学上均接近于禾谷镰孢菌复合种（Fusarium graminearum species complex）,而对其翻译延伸因子（EF-1α）序列分析发现,其均与亚洲镰孢菌聚在同一分支上,同源性达99%以上。因此,根据菌株形态学特征和EF-1α基因序列分析结果,将引致山黧豆根腐病的病原菌确定为亚洲镰孢菌（Fusarium asiaticum）。     

不同群落生境蒙古冰草种群株丛结构和叶片功能性状的变化

为探究蒙古冰草种群动态及叶功能性状在不同群落生境的变化，以宁夏盐池县荒漠草原蒙古冰草+草木樨状黄芪（MC）、蒙古冰草+老瓜头（ML）、蒙古冰草+牛枝子（MN）3种不同群落生境为研究对象，按丛径将蒙古冰草个体划分为Ⅰ级株丛（0~2.0 cm）、Ⅱ级株丛（2.1~4.0 cm）、Ⅲ级株丛（4.1~6.0 cm）、Ⅳ级株丛（6.1~8.0 cm）、Ⅴ级株丛（8.1~10.0 cm）和Ⅵ级株丛（>10.0 cm），研究了不同群落生境蒙古冰草种群的株丛结构和叶功能性状。结果表明：在蒙古冰草+草木樨状黄芪和蒙古冰草+牛枝子群落生境中，Ⅰ、Ⅱ级株丛较多，株丛密度较大，在蒙古冰草+老瓜头群落生境中，以Ⅲ、Ⅳ级株丛为主，株丛密度较小。蒙古冰草+草木樨状黄芪和蒙古冰草+老瓜头群落生境中蒙古冰草叶面积和比叶面积分别为4.68 cm²和41.2 cm²·g^-1、4.70 cm²和39.2 cm²·g^-1，显著高于蒙古冰草+牛枝子群落生境的3.38 cm²和27.7 cm²·g^-1。蒙古冰草株丛结构主要受土壤全钾的影响，而土壤水分、有机碳、全氮和全磷是影响叶功能性状的主要因子。由此，可通过改变种群数量特征和叶功能性状以适应自然生境的变化。     

铁杆蒿枯落物对四种豆科牧草的化感作用

研究了黄土区优势植物铁杆蒿（Artemisia gmelinii）枯落物对红豆草（Onobrychis viciaefolia）、沙打旺（Astragalus adsurgens）、百脉根（Lotusc corniculatus）和紫花苜蓿（Medicago sativa）等四种常见豆科牧草种子萌发和幼苗生理特性的化感效应。结果表明：铁杆蒿对这几种豆科牧草的发芽率和发芽势总体上均呈现“低质量浓度促进,高质量浓度抑制”的效应;紫花苜蓿综合化感效应在不同处理中均受到抑制,随着铁杆蒿枯落物含量的增加,抑制作用增强;但红豆草、百脉根和沙打旺的综合化感效应表现出低促高抑的趋势;红豆草和沙打旺在30 g铁杆蒿枯落物浓度时促进效应最强,百脉根在60 g时促进效应最强。以上结果初步表明在有铁杆蒿分布的草地不适合种植以及补播紫花苜蓿,而适当密度的铁杆蒿分布有助于种植以及补播红豆草、百脉根和沙打旺。