小蓬NeMT2基因的克隆及其植物表达载体的构建

该研究利用RACE ( Rapid amplification of cDNA ends)技术从小蓬中成功分离编码金属硫蛋白( Metal-lothionein,MT)的cDNA序列,命名为NeMT2,在GenBank中登录号为KT835290。该基因全长590 bp,开放阅读框为237 bp,编码78个氨基酸,编码的氨基酸序列中含有14个半胱氨酸残基( Cys,C),呈C-C,C-X-C,C-X-X-C排列,集中分布在肽链的N端和C端,基因编码蛋白的分子量为7.6036 kD,等电点为4.71。系统发育分析表明,小蓬金属硫蛋白NeMT2与藜科的海蓬子( AEF01492)和盐穗木( AHI62953)同源性最高,其次是甜菜( XP 010667708.1)。生物信息学分析表明,金属硫蛋白NeMT2无信号肽结构,属于非跨膜亲水性蛋白;疏水性分析表明,NeMT2蛋白的35~45个氨基酸之间有较强的疏水性,其中第41位Asp具最强的疏水性(1.444);结构预测分析该蛋白质二级结构的主要元件是无规则卷曲。通过RT-PCR对NeMT2基因的表达分析发现, NeMT2基因在铜矿区和非铜矿区的小蓬叶片中均有表达,但该基因在铜矿区小蓬叶片的表达量明显高于非铜矿区。将小蓬NeMT2基因定向克隆到植物表达载体pCAMBIA1300的35S 启动子下游,构建该基因的植物超表达载体pCAMBIA1300＋NeMT2。该研究结果为进一步研究该基因的功能和小蓬响应重金属胁迫的分子机制提供了一定基础。     

拉雅松遗传多样性及同域分布种间亲缘关系分析

拉雅松是广西西北部稀有的乡土用材、用脂树种,有较高的经济价值,但其遗传多样性状况及种间进化关系未知。利用SSR分子标记检测拉雅松群体遗传多样性,希望对该物种保护策略的制定提供参考依据。此外,鉴于该地区自然分布的松属树种仅有拉雅松、细叶云南松与马尾松三个种,试图利用SSR分子标记信息分析拉雅松与细叶云南松、马尾松的种间亲缘关系。结果表明:7对SSR引物在拉雅松群体共检测到14个等位基因。有效等位基因数为1.653,观测杂合度为0.577,期望杂合度为0.374,Shannon信息指数为0.540,Nei多样性指数为0.367,表明拉雅松具有较高的遗传多样性。拉雅松与马尾松遗传距离最近为0.0175,与3个细叶云南松群体距离较远,平均为0.0525。拉雅松与马尾松、细叶云南松平均共祖系数(Θ)分别为0.094、0.066,据此推测拉雅松可能与马尾松存在较近的亲缘关系。讨论了拉雅松的遗传多样性保护策略。     

Occurrence of Ochratoxin A-Producing Fungi in Commercial Corn Kernels in Argentina

Potentially ochratoxigenic Aspergillus and Penicillium species were identified and the natural occurrence of ochratoxin A (OTA) in corn kernels was evaluated. Likewise, the capacity to produce OTA by Aspergillus section Nigri and Circumdati was investigated. A total of 50 corn samples for human consumption was collected in the south of Córdoba Province. The surface-disinfected method for mycobiota determination was used. The OTA detection was performed by HPLC. OTA production was tested in strains belonging to section Nigri and Circumdati. Statistical analysis demonstrated that the specie A. flavus was isolated in higher frequency (p<0.01) from corn kernels in DRBC and DG18 media. The percentage of corn kernels contaminated by A. niger var. niger was similar in DRBC and DG18 media. The frequency of grains contaminated by A. flavus and A. niger var. awamori was higher than A. niger var. niger and A. japonicus var. japonicus (p<0.01) in DG18 media. The other potentially ochratoxigenic species, A. ochraceus, was isolated between 5% and 10% of the corn kernels in DG18 and DRBC media, respectively. The OTA producing species P. verrucosum was not isolated. All samples of corn were OTA negative (<1 ng g⁻¹). Thirty strains (25%) of the black Aspergillus were OTA producers. From four strains of A. ochraceus isolated, only one produced OTA. Due to the storage variable conditions could not be adequate in this substrate, the presence of ochratoxigenic strains of section Nigri and OTA needs to be evaluated for a longer time to establish the toxicological risk for human beings. The contamination of stored corn kernels with A. flavus and Aspergillus section Nigri was significant.     

Frequency of occurrence of mangrove fungi from the east coast of India

This paper describes the frequency of occurrence and biodiversity of fungi from mangroves of the Godavari and Krishna deltas, on the east coast of India. Seventy three species were identified from Godavari and 67 from the Krishna mangroves. Fifty five species were common to both sites, 18 were found only at Godavari and 12 at Krishna mangroves. Verruculina enaliawas found to be very frequent at both sites with a higher frequency of occurrence at Godavari. Eutypa bathurstensis was very frequent at Godavari but only frequent at Krishna. Cirrenalia pygmea and Cryptosphaeria mangrovei were frequent at the Godavari mangrove, but were recorded occasionally at Krishna. Decaying samples of Rhizophora and Avicennia were studied in detail. Forty three species were common to both hosts, while 22 species were recorded only from Avicennia and 20 only from Rhizophora. Verruculina enalia was the only very frequent fungus recorded on both hosts with a lower percentage occurrence (14.8%) on R. apiculata as compared to Avicennia spp. (24.3%). Eutypa bathurstensis was the next most frequent fungus on Avicennia, while Rhizophila marina was next most frequent on Rhizophora. Dactylospora haliotrepha which was recorded frequently on Rhizophorawas infrequent on Avicennia.     

Antagonistic actions of Pythium oligandrum and Trichoderma harzianum against phytopathogenic fungi (Fusarium oxysporum and Pythium ultimum var. ultimum)

Abstract

The possible biological control of damping-off fungi, Fusarium oxysporum and Pythium ultimum by Pythium oligandrum or Trichoderma harzianum was in vitro investigated. Results of comparing the antagonistic activity of P. oligandrum and T. harzianum in dual plates against the tested phytopathogens indicated different degrees of antagonism. After 12 days of incubation colony of the phytopathogenic fungus was completely overgrown by the antagonist, except for the interaction between T. harzianum and F. oxysporum which showed no overgrowth or any hyphal penetration by the antagonist. However, growth and proliferation of F. oxysporum colony was repressed. T. harzianum and P. oligandrum produced chitinase and β-1,3-glucanase when they were grown on liquid culture medium supplemented with chitin or fungal dried mycelium as a sole carbon source, and enzyme production was higher by T. harzianum comparing with P. oligandrum under the same condition. Fungal dried mycelium of F. oxysporum was the most selective carbon source for enzyme production, on the other hand, chitinase production was significant locked when P. ultimum dried mycelium was used as a carbon source. Production of volatile compounds by P. oligandrum or T. harzianum against F. oxysporum and P. ultimum was examined using the inverted plates method. F. oxysporum was inhibited by the antagonist volatile compounds and it is inhibited 100% by increasing the amount of inoculum size. Production of potential biocontrol agents provided with economically features and working under field conditions are recommended.     

Asexual reproductive organ-specific expression of the glyceraldehyde-3-phosphate dehydrogenase 2 gene of Pilobolus crystallinus

Pilobolus crystallinus has three putative glyceraldehyde-3-phosphate dehydrogenase (gapdh) genes (pcgapdh1, pcgapdh2 and pcgapdh3). The results of this study demonstrate that expression of pcgapdh2 was increased by irradiation and that this increased expression was correlated with the formation of asexual reproductive organs (trophocysts). Interestingly, expression of pcgapdh2 was restricted to trophocysts. The formation of trophocysts was likely promoted by light, and the expression of pcgapdh2 was increased as a result of trophocyst formation. This is the first report that shows the regulation of a gapdh gene in an organ-specific manner in fungi.     

Arbuscular mycorrhizas in coastal sand dunes of the Paraguaná Peninsula, Venezuela

Arbuscular mycorrhizal colonization was measured in the most abundant plant species of the Paraguaná Peninsula, northwestern Venezuela. These plant species included: Acacia tortuosa, Argusia gnaphalodes, Croton punctatus, Croton rhamnifolius, Egletes prostrata, Melochia tomentosa, Panicum vaginatum, Scaevola plumieri, Sporobolus virginicus, Suriana maritima, Leptothrium rigidum, and Fimbristylis cymosa. Mycorrhizal colonization was assessed using the Trouvelot et al. (1986) method that allows for simultaneous evaluation of frequency of colonization (%F), intensity of colonization (%M), and the proportion of arbuscules (%A) and vesicles (%V) present in the roots. Average frequency of colonization was 69%. The highest frequency of colonization was around 92% in C. rhamnifolius and A. tortuosa; in the other species, it varied from 49 to 86%. L. rigidum and F. cymosa were considered nonmycorrhizal because its colonization was very scarce and at all times appeared without arbuscules. Average intensity of colonization was 7%. The highest intensity of colonization was 18% in C. rhamnifolius. In the other species, it varied from 3 to 15%. Paspalum vaginatum, A. gnaphalodes, M. tomentosa, and S. maritima had their fungal structures tightly packed in modified little ovoid roots. In general, frequency of AM colonization was high and similar to those reported for other tropical ecosystems, whereas the intensity of AM colonization was low and similar to values obtained in analogous studies in disturbed ecosystems.     

A newly developed PCR-based method revealed distinct Fusobacterium nucleatum subspecies infection patterns in colorectal cancer

Fusobacterium nucleatum, which has four subspecies (nucleatum, animalis, vincentii and polymorphum), plays an important role in promoting colorectal cancer (CRC). However, as there is no efficient method of differentiating these subspecies in the context of a rich gut microbiota, the compositions in CRC remain largely unknown. In this study, a PCR-based differentiation method enabling profiling of F. nucleatum infection in CRC at the subspecies level was developed. Based on the analysis of 53 F. nucleatum genomes, we identified genetic markers specific to each subspecies and designed primers for the conserved sequences of those markers. The PCR performance of the primers was tested with F. nucleatum and non-nucleatum Fusobacterium strains, and complete consistence with taxonomy was achieved. Additionally, no non-specific amplification occurred when using human DNA. The method was then applied to faecal (n = 58) and fresh-frozen tumour tissue (n = 100) samples from CRC patients, and wide heterogeneity in F. nucleatum subspecies compositions in the gut microbiota among CRC patients was observed. Single-subspecies colonization was common, whereas coexistence of four subspecies was rare. Subspecies animalis was most prevalent, while nucleatum was not frequently detected. The results of this study contribute to our understanding of the pathogenicity of F. nucleatum at the subspecies level and the method developed has potential for clinical and epidemiological use.     

Expression of Apoptotic and Antioxidant Enzyme Genes in Sheep Oocytes and In Vitro Produced Embryos

The present study was to find out the expression pattern and relative expression level of apoptotic (Bcl2, Bax, Casp3, and PCNA) and antioxidant enzyme [(GPx, Cu/Zn-SOD (SOD1) and Mn-SOD (SOD2)] genes in sheep oocytes and developing embryos produced in vitro by conventional RT-PCR and real time qPCR, respectively. Different developmental stages of embryos were produced in vitro from oocytes collected from local slaughter house ovaries. RT-PCR amplicons showed expression of Bcl2 and PCNA in all stages except at morula. In contrast Bax and Casp3 were expressed in all stages. GPx and SOD1 were expressed in all stages but SOD2 was not expressed in 8–16 cells, although expressed in the remaining stages. The qPCR analysis reflected that Bcl2 expression was significantly (P < 0.05) downregulated in morula and maximum upregulated expression was observed in in vitro matured oocytes. Higher upregulated expression (P < 0.05) of Bax was in morula and downregulated expression was at 2-4 cells. Casp3 was significantly upregulated at 8–16 cells and downregulated in in vitro matured oocyte. PCNA expression was highest at blastocyst and least expression was at morula. GPx was expressed significantly highest in matured oocytes and least expression was at zygote. SOD1 was expressed significantly highest at 8–16 cells and least expression was at zygote. Expression of SOD2 was least among all the antioxidant enzymes but significantly higher expression of SOD2 was in immature oocyte; however, least expression was at 8–16 cells. It can be concluded from the study that the sheep embryos produced in vitro are highly sensitive to culture condition, which alters the expression level of apoptotic and antioxidant enzyme genes.     

迟缓爱德华氏菌Eta1-L-Gapdh融合蛋白在蓝藻中的表达

在蓝藻中表达迟缓爱德华氏菌Eta1-L-Gapdh融合蛋白。提取迟缓爱德华氏菌基因组DNA为模板,用PCR技术分别扩增两个已知具有较强免疫原性的基因eta1和gapdh,再采用重叠延伸PCR将这两个基因融合,获得目的融合基因eta1-L-gapdh。将目的基因连接到表达载体pRL489的两个Bam H I酶切位点之间构建表达载体,用质粒提取、PCR、酶切、测序等手段对表达载体进行验证。验证正确的表达载体通过三亲接合转化野生鱼腥藻PCC7120,用新霉素抗性筛选出转基因藻落,通过质粒提取和PCR验证转基因藻。用RT-PCR和Western-blot分别从转录水平和翻译水平对转基因藻中融合基因的表达进行了检测。结果表明,含目的基因的表达载体构建成功,目的基因在蓝藻中转录并表达蛋白,该蛋白在蓝藻中的表达量为2.46%。     

A survey of black scale,Saissetia oleae [Hom.: Coccidae] parasitoids [Hym.: Chalcidoidea] in southern California

A survey of black scaleSaissetia oleae (Olivier) parasitoids present in southern California was conducted between September 1987 and September 1989. From 308 collections of black scale-infested citrus, olive, and oleander twigs from 19 sites in southern California, 1,610 specimens were collected. Nine primary and six secondary parasitoid species were identified. Four primary species were abundant in southern California:Metaphycus bartletti Annecke & Mynhardt,M. helvolus (Compere),Scutellista caerulea (Fonscolombe) (=S. cyanea Motschulsky), andDiversinervus elegans Silvestri. The most common secondary parasitoids wereMarietta mexicana (Howard),Cheiloneurus noxius Compere, andTetrastichus minutus (Howard). In the coastal region of southern California,M. bartletti was the most abundant parasitoid, followed in order byD. elegans, S. Caerulea, andM. helvolus. In the intermediate and interior regions,M. helvolus was most abundant.D. elegans was second most abundant in the intermediate region, but was rare in the interior region.M. bartletti was second in abundance in the interior region and third in the intermediate region.      

Characterization of a broad range antibacterial substance from a new <Emphasis Type="Italic">Bacillus</Emphasis> species isolated from Amazon basin

A Bacillus sp. strain producing a bacteriocin-like substance was characterized by biochemical profiling and 16S rDNA sequencing. The phylogenetic analysis indicated that this strain has low sequence similarity with most Bacillus spp., suggesting a new species was isolated. The antimicrobial activity was detected starting at the exponential growth phase, and maximum activity was observed at stationary phase. The substance was inhibitory to a broad range of indicator strains, incluing pathogenic and food spoilage bacteria such as Listeria monocytogenes, B. cereus, Aeromonas hydrophila, Erwinia carotovora, Pasteurella haemolytica, Salmonella Gallinarum, among other. The antibacterial substance was stable over a wide pH range, but it was sensitive to pronase E and lipase. The antibacterial substance was bactericidal and bacteriolytic to L. monocytogenes and B. cereus at 160 AU ml⁻¹. The identification of a broad range bacteriocin-like inhibitory substance active against L. monocytogenes addresses an important aspect of food protection against pathogens and spoilage microorganisms.     

Interconversions of Chlorophylls a and b Synthesized from Exogenous Chlorophyllides a and b in Etiolated and Post-Etiolated Rye Seedlings

A comparative study of reciprocal conversions of chlorophylls a and b (Chl aand Chl b) in etiolated and post-etiolated rye seedlings (Secale cereale L.) was performed. The production of these pigments was initiated by infiltration of exogenous chlorophyllides a and b (Chlide a and b). It was shown that Chlide b, when infiltrated into etiolated rye seedlings, was esterified, producing Chl b. A major portion of Chl b (more than 80%) was transformed into Chl aduring long-term seedling dark exposure. The high rate of Chl b conversion into Chl a in the pool of pigments of exogenous origin was also observed during the lag-phase when there was no chlorophyll formation from endogenous precursors. The infiltration of Chlide a resulted in Chl a formation. The efficiency of its conversion into Chl b was low (about 1%) in the etiolated seedlings but increased during their greening. In the post-etiolated seedlings infiltrated with Chlide b, which were preliminary illuminated for 6–12 h, the Chl /Chl a ratio was almost similar in the pools of pigments synthesized from both exogenous and endogenous precursors. The rates of direct and reverse reactions responsible for the interconversion of Chl aand Chl b depended on the stage of the formation of the photosynthetic apparatus during greening of etiolated seedlings, when the particular structural components are formed in a definite sequence.     

Ontogeny of angiopoietin‐like protein 1, 2, 3, 4, 5, and 7 genes during chick embryonic development

Angiopoietin‐like proteins (ANGPTLs) are secreted proteins possessing an amino‐terminal coiled‐coil domain and a carboxyl‐terminal fibrinogen‐like domain and are known as angiogenic factors. Several members of ANGPTLs also regulate lipid metabolism independently of angiogenic effects, but most of their functions during vertebrate development are not demonstrated. To ascertain their developmental functions, we examined the expression patterns of Angptl1, 2, 3, 4, 5, and 7 orthologues during chick development using whole‐mount in situ hybridization. Angptl1 was first detected at embryonic day 3 (E3) in the somite. At E4, Angptl1 was expressed in somite‐derivatives and limb mesenchyme. Angptl2 was first detected at E3 in the hindbrain. At E4, Angptl2 was expressed in neuroepithelium of forebrain and hindbrain and partly in the heart. Angptl3 was first detected at E3 and continued to be expressed in the liver and yolk sac at E4. Angptl4 was first detected at E3 in the somites and liver. At E4, Angptl4 was also observed in the heart. Angptl5 was not detected in these developmental stages. Angptl7 was first detected at E3 in the ectoderm overlying the lenses of the eyes. At E4, Angptl7 was specifically expressed in cornea. These data suggest that each member of the ANGPTL family could be related to angiogenesis during various organogeneses of the developing chick embryo.     

Selective antimicrobial action of chitosan against spoilage yeasts in mixed culture fermentations

The effect of chitosan on Saccharomyces cerevisiae (the yeast that carries out alcohol fermentation), Brettanomyces bruxellensis and Brettanomyces intermedius (contaminants of alcohol fermentations), was investigated. The effect of chitosan was tested on each yeast, as well as on mixed cultivations of S. cerevisiae + B. bruxellensis and S. cerevisiae + B. intermedius. Chitosan enhanced the lag period of both strains of Brettanomyces (80 h for B. bruxellensis and 170 h for B. intermedius with 6 and 2 g/l chitosan, respectively). The growth rate of S. cerevisiae was inversely proportional to the chitosan concentration; the former was 50% when 6 g/l polysaccharide was used. Moreover, in mixed cultivations of S. cerevisiae and Brettanomyces strains, it was found that both B. bruxellensis and B. intermedius failed to grow while growth of S. cerevisiae was not affected (using 3 and 6 g/l chitosan, respectively). An interesting collateral result was that the presence of chitosan accelerated the consumption of glucose in the mixed cultivations (60 h instead of 120 h).     

Hybridization of DNA from methanogenic bacteria with nitrogenase structural genes (nifHDK)

Summary Using the Southern hybridization technique, homologies were examined between restricted DNA of four methanogenic bacteria (Methanobacterium ivanovi, Methanobacterium thermoautotrophicum, Methanococcus voltae, Methanosarcina barkeri) and the nif (nitrogen fixation) genes of Klebsiella pneumoniae and Anabaena strain 7120. With K. pneumoniae probes, no hybridization was observed with nifA, nifNE, and nifJ but positive results were obtained with the nifHDK genes coding for nitrogenase. Homology was detected, in the four strains, with K. pneumoniae and Anabaena nifH probes. In M. voltae and M. ivanovi, the homology found with nifH was estimated to be about 70% and a weaker hybridization was observed also with nifD and nifK. In M. voltae, the sequence homologous to nifH was found on a 3.0 kbp HindIII fragment and sequences homologous to nifD and nifK on a 3.8 kbp HindIII fragment. The 3.0 kbp fragment was cloned and the region homologous to nifH was localized more precisely. When this fragment was used as a probe against other DNAs, it behaved as a K. pneumoniae and Anabaena nifH probe. The results suggest that the structural genes for nitrogenase may be present in archaebacteria and raise interesting questions regarding their evolution.     

A molecular systematic study ofCathaya, a relic genus of thePinaceae in China

The systematic position ofCathaya, a relic genus of thePinaceae, was discussed based on therbcL gene sequence. The sequence data were analysed with PAUP and MEGA programs. The great genetic distance value betweenCathaya and any other genus of thePinaceae showed thatCathaya was a distinct and isolated genus. The most parsimonious Fitch tree and neighbor-joining tree showed thatCathaya was distantly related to the clade comprisingAbies, Keteleeria, Pseudolarix andTsuga, and a sister group relationship betweenCathaya andPinus was weakly supported.Pseudotsuga is closely related toLarix. In theAbies-Keteleeria-Pseudolarix-Tsuga clade,Abies has a close relationship toKeteleeria whilePseudolarix is relatively closely related toTsuga.     

Laboratory host range testing of Neomusotima conspurcatalis (Lepidoptera: Crambidae) – a potential biological control agent of the invasive weed,Old World climbing fern,Lygodium microphyllum (Lygodiaceae)

Old World climbing fern, Lygodium microphyllum, is a serious invasive weed in south Florida. Development of biological control is vital for sustainable management of L. microphyllum. Neomusotima conspurcatalis was discovered in Hong Kong in 1997 and was subsequently found causing feeding damage on L. microphyllum in much of its native range in Asia. Quarantine testing of N. conspurcatalis used 37 non-Lygodium fern species representative of New World genera of cultivated ferns and fern allies, one gymnosperm, three crop species, six Lygodium species, and the primary host L. microphyllum. No significant oviposition or feeding was observed on any of the 41 non-Lygodium species evaluated. Oviposition and feeding occurred on all Lygodium species, but amounts were low and usually significantly less than observed on L. microphyllum. The exception was L. japonicum, which was preferred as an oviposition host. Neomusotima conspurcatalis was only able to complete development on L. japonicum and L. palmatum, but survival on these species was only half that occurring on L. microphyllum. Neomusotima conspurcatalis is a Lygodium specialist. Lygodium japonicum is an invasive weed in the United States. Lygodium palmatum is restricted to areas of the United States where freezing winter temperatures would be lethal to N. conspurcatalis. It was concluded that N. conspurcatalis would pose no threat to native or cultivated plants in North America or the Caribbean and should be considered for biocontrol of L. microphyllum. A release petition was submitted in 2005. An USDA-APHIS release permit for N. conspurcatalis was issued in 2007.     

Life history adaptations and stress tolerance of four domestic species of Drosophila

Life history traits and stress tolerance were studied in four domestic species of Drosophila–D. melanogaster, D. simulans, D. auraria and D. immigrans– to understand how they adapt to their environments. In all species, larval weight approximately doubled in 1 day. The relative egg weight (egg weight : pupal weight) was smaller and the larval period was longer in D. immigrans than in the other three species. The pupal period was the longest in D. auraria. However, the adaptive significance of these differences in larval and pupal periods was not clear. The pupal case was generally thicker in the larger species, probably to support the larger pupal body. The start of oviposition was earliest and reproductive effort was greatest in female D. simulans, followed by female D. melanogaster. In contrast, starvation tolerance and the increase in bodyweight after eclosion was greater in D. immigrans and D. auraria than in the other two species. Pupal desiccation tolerance was greatest in D. melanogaster and lowest in D. auraria, and the less tolerant species seemed to select more humid sites for pupation. Adult tolerance to desiccation was greatest in D. melanogaster and lowest in D. simulans. In contrast, adult cold tolerance was greater in D. auraria and adult heat tolerance was lower in D. immigrans than in the other species. These differences in life history traits and stress tolerance represent the Drosophila species differential adaptations, and are assumed to allow coexistence of the species.     

Molecular cloning of thentrA gene of the broad host-rangeRhizobium sp. NGR234, and phenotypes of a site-directed mutant

Summary The clonedntrA (rpoN) gene andntrA mutants ofRhizobium meliloti were used to isolate the homologous gene from the broad-host rangeRhizobium sp. NGR234 by hybridization and interspecies complementation. The NGR234 locus was analyzed by deletion and insertional mutagenesis. A site-directedntrA mutant, NGR234rn1, was made with an interposon, GmI, and its phenotype was examined ex planta and in symbiosis. NGR234rn1 formed Fix⁻ nodules on six genera tested from among its legume hosts, including both indeterminate and determinate nodule-type plants. Formation of nodules onMacroptilium was delayed, and expression of anR. meliloti nodABC-lacZ fusion was reduced by the mutant allele.