GUS基因标记法对慢生花生根瘤菌竞争结瘤和接种效果的研究

应用GUS基因标记技术,可简便、快速、准确、原位、直观地确定标记花生根瘤菌株形成的根瘤,从而方便地研究标记菌株与土著根瘤菌的竞争结瘤能力。无氮水培试验表明,标记菌株gusA4-5、gusA2-9分别与土著菌混和接种占瘤率为71.4%、77.0%。盆栽试验表明,接种供试菌株Spr4-5、Spr2-9占瘤率分别为57.9%、63.0%,比对照极显著增产52.5%、22.7%;接种Spr4-5比Spr2-9极显著增产24.2%。初步说明两个供试菌株的竞争结瘤力比土著根瘤菌强,菌株Spr2-9强于Spr4-5;Spr4-5比Spr2-9有效性高,是结瘤适量,竞争结瘤能力强的高效菌株。     

石灰性紫色土施铁肥与接种根瘤菌对花生-根瘤菌共生固氮作用的影响

以缺铁的石灰性紫色土为供试土壤进行盆栽实验,选用三株慢生型花生根瘤菌Spr3 5、Spr3 7、Spr4 5及gusA和celB标记的菌株gusA3 5、gusA3 7、gusA4 5、celB3 5、celB3 7、celB4 5接种天府9号花生。通过标记根瘤菌形成的根瘤能与检测试剂产生颜色反应的特征,检测施铁肥及施不同浓度的铁肥对花生 根瘤菌有效性和竞争性的影响。结果发现:缺铁的石灰性紫色土上单施铁肥、单接种根瘤菌、接种根瘤菌配施铁肥均能促进花生与根瘤菌的共生固氮效应和竞争结瘤能力,但接种根瘤菌配施铁肥的效果最好,单接种根瘤菌的效果次之,单施铁肥的效果差。喷施0 .2 %硫酸亚铁溶液的效果比0 .3%的好。植株全氮含量和叶绿素含量都是指示共生固氮效应的重要指标,与花生产量间存在极显著的相关性,相关系数分别为0 .76 3和0 .795。gusA和celB两种标记方法检测的结果基本一致,两种标记根瘤菌的平均占瘤率分别为79.6 4 %、75 .6 2 %、74 .4 1%。供试菌株中Spr4 5的有效性和竞争性最强,Spr3 7次之,Spr3 5最差     

根瘤菌中的nodD基因及其与弗兰克氏菌（Frankia）中的类nodD基因的功能互补

根瘤菌可在几种豆科植物上成瘤,结瘤基因（ｎｏｄ基因）赋予这些细菌以寄主专一性的方式诱导瘤的形成。目前,已被鉴定的三类主要结瘤基因是：＂公共＂结瘤基因,如ｎｏｄＡＢＣ,这类基因是结瘤不可缺少的,并具有高度的结构和功能上的保守性;另一类是寄主专一性基因,这类基因具有种属或菌株的专一性,它们决定了细菌的寄主范围;最后一类是结瘤基因Ｄ（ｎｏｄＤ）,这类基因编码一类调节蛋白,并和植物分泌的酚类化合物一道起动其他结瘤操纵子的转录。一些根瘤菌种属只有一个拷贝的ｎｏｄＤ基因,而另一些则可拥有三个拷贝之多。苜蓿根瘤菌（Ｒ．ｍｅｌｉｌｏｔｉ）中存在三个拷贝的ｎｏｄＤ基因,其中的任何一个发生突变都以依赖于寄主的方式影响结瘤效果。在慢生型大豆根瘤菌（Ｂ．ｊａｐｏｎｉｃｕｍ）中,ｎｏｄＤ１突变菌株还可在大豆或其他豆科植物寄主上结瘤,只是稍往后推迟而已,ｎｏｄＤ２基因对ｎｏｄＹＡＢＣＳＵＩＪ操纵子诱导的影响却很小,两个ｎｏｄＤ拷贝突变后尚表现出结瘤活性。在只有一个ｎｏｄＤ基因拷贝的根瘤菌种属中,如豌豆根瘤菌（Ｒ．ｌｅｇｕｍｉｎｏｓａｒｕｍ）和茎瘤固氮菌（Ａ．ｃａｕｌｉｎｏｄａｎｓ）中,ｎｏｄＤ突变后会导致菌株丧失结瘤能力。一般说来,不     

马占相思根瘤菌结瘤固氮特性的分析

相思属（Acacia）树种是速生的热带、亚热带豆科植物。对现在大面积种植的相思树品种——马占相思的根瘤菌进行分离和筛选，获得菌株若干株，对它们的结瘤固氮特性和对马占相思、大叶相思及厚荚相思生长的影响等进行了初步研究。结果表明。无氮条件下接种马占相思根瘤菌，能显著促进这3个树种的株重、株高和生物量的增长；苗圃接种马占相思根瘤菌。马占相思苗高增加27．6％，地径增加14．8％，植株鲜重增加32．6％，结瘤数增加83．8％。     

抗药性根瘤菌基因工程菌的构建与应用

用三亲本杂交的方法将外源抗药性质粒导入到高效固氮的根瘤菌 Tal377中,外源质粒在 Tal377中能正常表达,但不影响其原有的固氮结瘤能力。利用此抗药性根瘤菌所携带的抗药性,在接种此根瘤菌的同时结合施加一定浓度的抗菌素,抗药性根瘤菌与出发菌株相比表现出一定程度地提高其结瘤固氮能力和占瘤率。     

抗春雷霉素大豆根瘤菌突变株的诱变及其生物固氮特性的分析

根瘤菌与豆科植物共生可以固定大量的氮。根瘤菌剂接种豆科作物是一项普遍推广应用、有效的农业技术。但由于大量土著根瘤菌的存在，产生竞争障碍，降低了接种菌剂的占瘤率。大多数的土著根瘤菌对春雷霉素敏感，因此接种抗春雷霉素的高效结瘤固氮根瘤菌，并用春雷霉素处理种子，可抑制土著根瘤菌，提高接种菌剂的占瘤率，从而达到高结瘤、高固氮和提高产量的目的。本文将探讨诱变对根瘤菌抗春雷霉素突变的作用，并对获得的抗性突变株的生物固氮特性进行分析。     

成都平原蚕豆高效根瘤菌的筛选及其促生功能初步评价

[目的]筛选适用于成都平原的高效广谱蚕豆根瘤菌,并对其相关促生功能进行初步评价,为成都平原高效蚕豆根瘤菌剂的研制与应用提供科学依据。[方法]供试6株根瘤菌由课题组前期分离自成都平原,其与四川主栽蚕豆‘大白蚕豆’匹配良好,采用常规方法测定了这6个菌株分泌生长素及溶磷能力。菌株与蚕豆品种匹配试验采用低氮砂培法,供试蚕豆品种为成都平原主栽品种‘成胡14’、‘成胡15’;两个品种的蚕豆种子播种后,分别接种6个菌株,以不接种为对照(CK),光照(控温22~25℃、光照强度2800 lx左右、日照时间14 h)下培养41天后收获,测定植株生物量和根瘤数。然后,对匹配性试验筛到的两株高效广谱根瘤菌进行田间验证,供试蚕豆品种为成胡15,将2个根瘤菌制备的菌剂(活菌数5.0×10^8 CFU/g以上,载体为泥炭)进行拌种,以不接菌处理的灭菌泥炭为对照。在盛花期(生育期105 d)采样测定株高、根瘤数、地上部分植株干重;收获期(生育期200 d)采样测产;测定两个时期植株样品氮、磷、钾含量。盛花期采用BOX-PCR分子标记法测定接种根瘤菌占瘤率,同时提取接种菌株SCAUf73、SCAUf76的总DNA,比较接种菌株及相应根瘤类菌体根瘤菌DNA的BOX-PCR分子指纹图谱。用多位点基因序列分析法对田间验证的优良菌株SCAU73进行分类地位研究。[结果]1)通过匹配性砂培试验,筛选到2株与2个成都平原主栽蚕豆品种均高效匹配的根瘤菌SCAUf73、SCAUf76。SCAUf76、SCAUf73能使‘成胡14’、‘成胡15’植株干重较CK显著增加40.5%~61.6%。2)通过两株菌田间接种试验发现,接种SCAUf76处理的蚕豆产量与CK差异不显著;接种SCAUf73处理蚕豆植株干重、全氮含量等指标均高于CK,籽粒鲜产比CK显著增加25.0%,并显著高于SCAUf76,其占瘤率达到33%。3)多位点基因序列分析表明,SCAUf73可能是Rhizobium的一个新类群。4)促生性试验表明,6株菌都能分泌生长素(IAA),最大分泌量为21.0 mg/L(SCAUf76);供试菌株的溶磷能力不明显。[结论]从成都平原上筛选的6个菌株中,SCAUf73具有分泌IAA能力,与蚕豆接种后,占瘤率达33%,可显著促进蚕豆氮素吸收积累,提高蚕豆籽粒产量。与成都平原的主栽蚕豆品种匹配的高效广谱根瘤菌SCAUf73,适用于成都平原的蚕豆生产。     

接种耐酸根瘤菌和施钙对酸性土上紫花苜蓿生长的影响

采用裂区设计法研究了重庆缙云山酸性土上施钙与接种耐酸根瘤菌对紫花苜蓿生长和品质的影响。结果发现:接种耐酸苜蓿根瘤菌对苜蓿植株瘤重、根鲜重、株高、植株上部鲜重、全氮含量和酸性土壤中根瘤菌数量的提高影响显著;施Ca2+ 5 mmol/L与10 mmol/L处理与不施Ca2+处理相比,能显著增加植株根鲜重、株高和地上部鲜重,也能显著增加瘤重;瘤重与植株地上部鲜重、根鲜重和株高呈极显著相关关系,与植株全氮含量呈显著相关关系,说明良好的结瘤性能能够提高酸性土上紫花苜蓿的产量和品质。     

Mo与花生根瘤菌的复合菌剂对盛花期花生生长的影响

通过盆栽试验分析了"Mo+花生根瘤菌Spr2-9"复合菌剂对花生盛花期生长的影响.结果表明:(1)单接种Spr2-9(R1)能明显或显著增加植株干重、叶绿素和全氮量,分别比相应对照增加10.0%,14.6%,37.0%.(2)复合菌剂处理(R2,R3,R4)的占瘤率随钼浓度增大而增大,平均单瘤重随钼浓度增大显著降低,而总瘤数、全氮、叶绿素含量、植株干重随钼浓度的变化不显著.(3)钼能显著促进盛花期无菌处理(2,3,4)植株和根瘤的生长,平均单瘤重、叶绿素含量、植株干重、全氮量等随钼浓度增大显著增高,但对总根瘤数的影响不大.(4)等钼量的复合菌剂和无菌处理间,除无菌处理的单瘤重显著或明显高于复合菌剂外,其它都是复合菌剂处理高于无菌处理.表明供试根瘤菌是高效菌株,"Mo+供试根瘤菌"的复合菌剂对竞争结瘤、根瘤及植株生长有明显作用.     

酸性紫色土上紫花苜蓿的结瘤性能与养分吸收

盆栽试验表明,在pH.5.45的酸性紫色土接种苜蓿根瘤菌,可显著提高紫花苜蓿地上部和地下部生物量。根瘤菌不同株系的结瘤性能和对宿主生长的影响存在显著差异,且不同紫花苜蓿品种的结瘤性能也存在显著差异。结瘤性能良好的菌株显著提高地上部氮、磷和钙的含量,而降低地上部钾含量和铝含量。紫花苜蓿地上部的氮、磷、钾和钙含量高于地下部,镁含量差异不大,而铝含量在接种后地下部含量高于地上部。相关分析表明,紫花苜蓿根瘤重和根瘤数与地上部生物量和地上部氮含量之间存在显著正相关关系;而地下部铝含量与地上部生物量、结瘤性能和植株氮含量呈显著负相关关系。说明酸性土壤良好的结瘤性能可提高生物固氮效率、促进紫花苜蓿生长,但根系铝的过量吸收影响紫花苜蓿的生长、结瘤与氮素吸收。     

固氮芽孢杆菌N3的筛选鉴定及其对二月兰的促生效果

从山东泰安农田土壤中筛选获得1株固氮能力强的菌株N3,通过形态观察、生理生化特征以及16S rDNA基因序列分析,确定为巨大芽孢杆菌属(Bacillus megaterium),该菌株固氮酶活性达C2H428.33 nmol/(h·ml)。温室条件下进行二月兰盆栽试验,设置不接菌对照(CK)、接种巨大芽孢杆菌N3、接种产吲哚乙酸(IAA)菌CDL29和复合接种菌株N3+CDL29四个处理,探讨了接种不同植物促生菌对二月兰的促生效果。结果表明:接种植物促生菌均促进二月兰的生长,且单一接种固氮菌株N3的促生效果最好,其显著提高二月兰地上部氮、磷和地下部氮、钾的含量,相比对照,增幅分别为33.03%、55.56%和29.87%、51.11%,其他处理也有提高二月兰养分含量的趋势,但并不显著;单一接种菌株N3,土壤有机质、全氮和速效养分含量显著提高;接种植物促生菌均提高土壤微生物生物量碳(MBC)和氮(MBN)的含量,单一接种固氮菌株N3提升效果最佳,接种产IAA菌株CDL29和复合接种次之。此外,接种固氮菌株N3能够显著提升土壤固氮酶活性,接种菌株CDL29对土壤固氮酶活性没有显著影响。因此,单一接种固氮菌株N3在促进二月兰生长、养分含量提高及土壤理化和生物学性质改良上都具有显著的效果。     

不同氮效率木薯品种根系形态、构型及氮吸收动力学特征

【目的】比较分析低氮 (N) 条件下不同氮效率木薯品种的根系形态、构型及吸收动力学变化,以阐明木薯氮高效吸收机制,为指导木薯生产和木薯品种选育提供理论基础。【方法】于2015年在广西大学国家重点实验室温室大棚内进行了试验。盆栽试验采用双因素 (品种 × 氮水平) 区组设计。供试木薯品种包括氮高效品种华南10号 (SC10) 与氮低效品种华南205(SC205)。氮水平包括不施氮 (N0) 和施N 55.2 mg/kg土 (N1)。每盆装10 kg土,种植1株幼苗。木薯出苗60天后,取出并洗净根系,利用根系扫描仪EPSON2000进行根系图像采集,利用WinRHIZO PRO根系分析软件分析图片,获得根系形态指标。将整株植株分成根、茎、叶三个部分,测量干重和氮含量。根系分层试验在大型根系观测系统中进行。吸收动力学试验采用改进常规耗竭法,并比较分析了木薯根系形态、根系构型特征及硝态氮吸收动力学参数差异。【结果】N1和N0条件下,氮高效品种SC10生物量和氮素积累量均显著高于氮低效品种SC205(P < 0.05)。N0条件下,SC10的整株生物量降幅为37.4%,SC205的降幅为69.4%,品种SC10的降幅显著低于SC205 (P < 0.05);SC10的根、茎、叶和全株氮积累量均显著高于SC205,全株氮积累量为SC205的152%。与N1相比,N0处理的木薯品种SC10总根长、根系表面积和细根根长的降幅分别为11.0%、10.0%和20.4%,SC205的降幅分别高达35.9%、27.7%和50.2%,两个品种下降幅度差异显著 (P < 0.05)。低氮诱导木薯根系分布下移,SC10根系呈上宽下窄三角形,最深可达180 cm土层;SC205根系呈椭圆形,最深达130cm土层。氮素吸收动力学试验结果发现SC10、SC205的Km分别为3.27和7.87 mmol/L,表明SC10根系对NO₃–的亲和性更高。【结论】氮高效品种SC10的根系对硝态氮的亲和力 (Km) 几乎是氮低效品种SC205的两倍。在氮素胁迫条件下,氮高效品种可形成优于氮低效品种的根系构型,特别是根系的总根长、根系表面积和细根根长的下降幅度显著小于氮低效品种,是有效缓解氮胁迫的重要原因。     

砂姜黑土中兼具促生功能的纤维素降解菌的筛选及其应用

为实现砂姜黑土地区秸秆资源化利用以及作物增产，本研究从该区小麦-玉米轮作土壤中筛选具有促生功能的纤维素降解菌株。通过羧甲基纤维素酶活(CMC)与吲哚乙酸(IAA)分泌量测定，筛选出1株具有高效降解纤维素的促生菌株。经形态学和分子生物学鉴定，该菌株为普沙根瘤菌(Rhizobium pusense)，命名为X2。小麦秸秆降解和玉米盆栽初步试验结果显示，X2菌株经液态发酵15d后小麦秸秆降解率为16.1%，较对照显著提升65.4%；接种该菌株后盆栽土壤中碱解氮含量较对照显著提高了72.7%，植株根系的平均直径、表面积以及地上部干物质量显著增加了22.0%、28.6%和33.3%。为进一步提高该菌株的活性，采用单因素试验对菌株的培养条件进行优化，结果表明，X2菌株在装液量为25 mL/250 mL、氮源为酵母粉、pH为6.0时生长和产IAA能力最优，产酶的最适pH为5.0。综上，所筛菌株X2具有秸秆降解和玉米促生能力，可在砂姜黑土地区为秸秆资源化利用提供新的微生物资源。     

土壤中大豆根瘤菌之间竞争结瘤的研究——Ⅲ.接种菌量对大豆生长的影响

本文研究了大豆根瘸菌PRC005的接菌量对大豆生长的影响.田间试验结果表明:接菌量在播种后40天和60天没有显著增加根瘤数、根瘤干重、地上部植株干重和植株含氮量.施氮肥处理和较高接菌量处理之间的大豆种子产量差异不显著,与不接菌对照处理相比,施氮肥和较高接菌量两个处理的种子产量显著增加.施氮肥处理没有使植株含氮量增加,并且还妨碍了大豆的结瘸作用.室内盆栽试验结果表明:只有当接菌量高于土著菌数1200倍时,才能显著地提高大豆的结瘤数和植株干重.     

不同施氮方式对甘蔗氮肥效率及氮素去向的影响

以新台糖22号(ROC22)为试材,通过网室盆栽试验方法,在总施氮量(15N标记尿素5g/盆,相当于450kg·hm-2)相同条件下,研究了全部基施(T1)、50%基施+50%在分蘖期追施(T2)和30%基肥+30%在分蘖期追施+40%在伸长期追施(T3)3种施氮方式对甘蔗氮肥效率与氮素去向的影响。结果表明:甘蔗吸收的氮素约18%～29%来源于当季施用的尿素氮,71%～82%来自土壤和种茎氮;氮肥利用率为21.0%～34.52%,残留率为37.61%～44.13%,有21.35%～41.39%的氮素损失。3种施氮方式下,氮肥残留在0～20cm土层较多,在20～40cm残留较少。随氮肥施用时间后移,甘蔗吸收的氮素、来源于肥料氮素的比率、氮肥利用率、氮肥残留率、蔗茎产量及产糖量明显增加,而氮肥损失率显著下降,蔗茎的氮素分配率和蔗糖分积累略呈下降趋势,同时氮素在0～20cm土层的残留呈上升趋势,在20～40cm土层呈下降趋势。从经济效益和环境效益考虑,T3施氮方式的效果较为理想。     

Forage Production,Nitrogen Fixation,and Soil N Accumulation of White Clover in the Hill Farming System of Azad Jammu and Kashmir

Quantitative measurements of plant growth characteristics, forage production, nitrogen (N) fixation, and soil N accumulation by white clover were determined in a field experiment at the subhumid hilly region of Rawalakot, Azad Jammu and Kashmir (AJK). Three indigenous and two exotic ecotypes of white clover were used in the study. Indigenous ecotypes were collected from three different locations (i.e., Tollipir, Banjosa, and Rawalakot), whereas exotic ecotypes (NuSiral and Irrigation) were collected from New South Wales Agricultural Research and Advisory Station, Australia. Data were collected for two seasons (spring 2004–autumn 2004). Total average values for height, number of stolons, length of stolons, number of leaves, and leaf area were 13–50 cm, 9–20, 2–4 cm, 23–81, and 7–16 cm², respectively. The morphological characteristics of exotic ecotypes were significantly higher than the indigenous ecotypes, and the percentage increase in different plant characteristics was +6% to 214%. Total herbage dry‐matter yield (DMY) in the indigenous and exotic ecotypes varied between 0.5–2.3 and 3.6–4 Mg ha^?1, respectively. All the ecotypes showed substantial nodulation potential, and the number of nodules in plant roots ranged from 65 to 119, confirming the presence of indigenous Rhizobium population in the soil. The N contents of harvested herbage of white clover were 2.3–3.0% compared to 0.85% in the grass, and the estimated rates of N₂ fixation were 26 kg N ha^?1 in the indigenous to 79 kg N ha^?1 in the exotic ecotypes. Amount of N₂ fixed was strongly correlated with DMY, suggesting that crop DM can be used as an indicator of N₂ fixation in white clover. Protein content of white clover was 14–19%, compared to 5% in the indigenous grass species. Total organic carbon (C) and N in control soil were 8.5 and 0.75 g kg^?1, which increased significantly to 13.1 and 0.93 g kg^?1 in soil under white clover. It is concluded that white clover has substantial potential for growth and establishment in the subhumid hilly regions and can be used to recuperate degraded soils because of its ability to sustain high level of pasture production and increase the N status of soil. These benefits could be of particular use for small‐scale resource‐poor farmers.     

Nodulation and competitiveness of gusA-marked Bradyrhizobium japonicum A1017 in soybean

Attempts to improve the symbiotic nitrogen fixation with effective (Brady) rhizobium strains do not always succeed under field conditions due to the lower nodulation competitiveness of the introduced strains than that of the indigenous rhizobia (Triplett and Sadowsky 1992). An introduced strain needs to be marked for monitoring its nodule occupancy under competitive nodulation conditions.     

一株产生氨氮和亚硝酸盐氮菌的筛选鉴定及特性研究

从4个草鱼池塘中分离和定性筛选获得29株能够产生氨氮和亚硝酸盐氮的菌株。通过对编号为C95的菌株进行菌落形态学观察和16S rDNA序列分析,表明该菌株为革兰氏阴性杆状菌,与寡养单胞菌属（Stenotrophomonas sp.）的同源性达98%。采用单因素多水平试验对菌株的产氨氮和产亚硝酸盐氮特性进行研究发现：（1）氮源、碳源、温度和摇床转速都能显著影响菌株的生长及产生氨氮和亚硝酸盐氮的含量,但pH（5～9）对其无显著影响（P〉0.05）;（2）该菌株生长及产生氨氮和亚硝酸盐氮最适宜的培养基以及培养条件为：LB、pH 5～9、25℃、150 r.min-1。由C95作为指示菌株筛选得到SC01、SC07两株（2/33）去除氨氮和亚硝酸盐氮效果较好的菌株。因此,C95可作为筛选具有降氨氮和亚硝酸盐氮功能的有益菌的指示菌株。     

Symbiotic and genomic diversity of ‘cowpea’ bradyrhizobia from soils in Botswana and South Africa

Strain CB756 is usually an effective competitor against indigenous bradyrhizobia for nodulation of peanut in South Africa. Recently, inoculation of peanut and cowpea with CB756 in loamy sand soils of Botswana or a sandy clay loam in South Africa proved unsuccessful, achieving <2% nodule occupancy. A survey of ‘cowpea’ bradyrhizobia from five soils in Botswana and one in South Africa showed that many were effective in ability to fix N₂ on peanut and cowpea. However, 15 isolates from Good Hope, Botswana were all effective on cowpea but ineffective on peanut, three failing to nodulate the latter. Selected cowpea isolates were significantly more competitive than CB756 for nodulation of cowpea in Leonard jars, but four were unsuccessful when inoculated at Roodeplaat, South Africa. When strain CB756 and two isolates were inoculated in pots containing Roodeplaat soil, at a 4:1 inoculant to soil bradyrhizobia ratio, their average nodule occupancy was 8% on cowpea compared to 40% on peanut. Significant differences in strain nodule occupancy were not detected on either cowpea or peanut. In contrast, nodule occupancy in loamy sand from Good Hope, Botswana, inoculated at a 40:1 inoculant to soil bradyrhizobia ratio, was 22.4% on cowpea and only 6.8% on peanut. In Good Hope soil, strain CB756 was the weakest competitor on cowpea but strain differences were insignificant on peanut. Whereas the Good Hope soil population was effective on cowpea, it was ineffective on peanut. DNA fingerprinting showed that isolates from Gaborone, Francistown and Roodeplaat contained several different genotypes, whereas those from Good Hope, Rasesa and Maun were more homogeneous. The dominance at Good Hope of genotypes effective on cowpea but ineffective on peanut emphasises the value of assessing the symbiotic capabilities and structures of indigenous populations.     

Competitive abilities of common field isolates and a commercial strain of Rhizobium leguminosarum bv. trifolii for clover nodule occupancy

We previously reported that commercial Rhizobium leguminosarum bv. trifolii inoculants failed to outcompete naturalized strains for nodule occupation of clover sown into an alkaline soil [Aust. J. Agric. Res. 53 (2002) 1019]. Two field isolates that dominated nodule occupancy at the field site were labeled with a PnifH-gusA marker. Marked strains were chosen on the basis that they were equally competitive and fixed similar amounts of nitrogen in comparison to their parental strain. The minitransposon insertions were cloned and sequence analysis revealed that neither lesion disrupted the integrity of any known gene. The marked strains were then used to follow nodule occupancy of Trifolium alexandrinum in competition against the commercial inoculant TA1 under a range of experimental conditions. In co-inoculation experiments in sand-vermiculite, TA1 outcompeted each marked field isolate for nodule occupancy. However, using TA1-inoculated seed sown into alkaline soil containing a marked field strain, it was demonstrated that by increasing the cell number of marked rhizobia in the soil and reducing the cell number of the commercial inoculant, the proportion of nodules occupied by TA1 was reduced. These studies indicate that the ability of the field isolates to dominate nodule occupancy in the alkaline field soils was most likely caused by poor commercial inoculant survival providing the advantage for naturalized soil rhizobia to initiate nodulation.