Development of transgenic imazapyr-tolerant cowpea (Vigna unguiculata)

Key message

Here we present the development of cowpea lines tolerant to a herbicide from imidazoline class (imazapyr). Plants presented tolerance to fourfold the commercial recommended dose for weed control.

Abstract

Cowpea is one of the most important and widely cultivated legumes in many parts of the world. Its cultivation is drastically affected by weeds, causing damages during growth and development of plants, competing for light, nutrients and water. Consequently, weed control is critical, especially using no-tillage farming systems. In tropical regions, no-till farming is much easier with the use of herbicides to control weeds. This study was conducted to evaluate the possibility of obtaining transgenic cowpea plants resistant to imidazolinone, which would facilitate weed control during the summer season. The biolistic process was used to insert a mutated acetohydroxyacid synthase coding gene (Atahas) which confers tolerance to imazapyr. The transgene integration was confirmed by Southern blot analysis. Out of ten lines tested for tolerance to 100 g ha^?1 imazapyr, eight presented some tolerance. One line (named 59) revealed high herbicide tolerance and developmental growth comparable to non-transgenic plants. This line was further tested for tolerance to higher herbicide concentrations and presented tolerance to 400 g ha^?1 imazapyr (fourfold the commercial recommended dose) with no visible symptoms. Line 59 will be the foundation for generating imidazolinone-tolerant cowpea varieties, which will facilitate cultivation of this crop in large areas.     

红壤坡地杂草群落VA菌根真菌的宿主物种调查

本研究调查了红壤坡地幼龄果园杂草群落的物种多样性以及主要物种被VA菌根真菌的侵染率和侵染强度,分析了VA菌根真菌侵染与根际土壤磷系水平的关系。研究区域幼龄果园杂草群落共有杂草96种,分属27科。对17个科的39个物种的调查发现,所调查的物种均不同程度上被VA真菌所侵染,但科与科之间存在显著差异,同一科的不同种之间也差异明显。相关分析表明,VA菌根真菌对不同毁草种 的侵染率和侵染强度与土壤磷素水平关系不甚密切,VA菌根真菌强度可能主要决定于宿主植物特性和VA菌根真菌的选择。因此,在不影响农业生产的前提下,尽可能保持杂草的多样性将有利于VA菌根真菌的生存。     

Susceptibility of some weed species to Pepino mosaic virus (PepMV)

The aim of our investigations was to study the susceptibility of 22 weed species to Pepino mosaic virus (PepMV). Seven plants of each species were mechanically inoculated at 4-6 leaf stage with PepMV in a vector free virological glasshouse. Inoculated plants were tested on the basis of symptoms, by DAS ELISA serological method and back inoculation. Among the 22 weed species, only some Solanum ones (S. aethiopicum, S. dulcamara, S. luteum, S. nigrum) were susceptible to PepMV infection. All of them--except S. aethiopicum--belong to Hungarian weed flora, therefore these weeds--occurring mainly in tomato ecosystems--may play important role in the epidemiology of PepMV. Other 18 weed species seemed to be resistant to PepMV, and on the basis of back inoculation not even latent infection has been observed.     

Identification of potential hosts plants of Cowpea aphid‐borne mosaic virus

Among the major pathogens affecting passion fruit orchards, the cowpea aphid‐borne mosaic virus (CABMV), also known as the fruit‐hardening virus, has gained prominence owing to its role in the drastic reduction in fruit production in yellow passion fruit orchards (Passiflora edulis f. flavicarpa) from the second year of cultivation. To mitigate the damage, several regions adopt the annual planting system where a sanitary void is maintained from August to September. However, the virus is believed to remain dormant in weeds. This study aimed to identify potential weed hosts of CABMV. The study was conducted with a randomized design with four replications in Londrina, PR. Twenty‐eight weed species were tested, and a sample of yellow passion fruit leaves symptomatic for CABMV infection was used as the virus inoculum source. Mechanical inoculation was performed using the extract from the symptomatic plant. Symptoms were visually evaluated every 3 days. For molecular confirmation, total RNA was extracted, followed by RT‐PCR with CABMV‐specific oligonucleotides, reinoculation in passion fruit plants and sequencing. CABMV infection was observed in southern sandbur (Cenchrus echinatus), Siberian motherwort (Leonurus sibiricus), showy rattlepod (Crotalaria spectabilis) and yellow passion fruit (Passiflora edulis). The CABMV‐positive weed species extract was able to infect yellow passion fruit plant when a fresh mechanical inoculation was performed. Showy rattlepod (Crotalaria spectabilis) was the only weed species to exhibit observable symptoms of CABMV. C. echinatus, L. sibiricus and C. spectabilis act as a source of CABMV inoculum.     

Mobilization of a Sym plasmid from a fast-growing cowpea Rhizobium strain.

A large Sym plasmid from a fast-growing cowpea Rhizobium species was made mobilizable by cointegration with plasmid pSUP1011, which carries the oriT region of RP4. This mobilizable Sym plasmid was transferred to a number of Rhizobium strains, in which nodulation and nitrogen fixation functions for symbiosis with plants of the cowpea group were expressed.     

Phylogeny of bradyrhizobia from Chinese cowpea miscellany inferred from 16S rRNA, atpD, glnII, and 16S-23S intergenic spacer sequences

The cowpea (Vigna unguiculata L.), peanut (Arachis hypogaea L.), and mung bean (Vigna radiata L.) belong to a group of plants known as the "cowpea miscellany" plants, which are widely cultivated throughout the tropic and subtropical zones of Africa and Asia. However, the phylogeny of the rhizobial strains that nodulate these plants is poorly understood. Previous studies have isolated a diversity of rhizobial strains from cowpea miscellany hosts and have suggested that, phylogenetically, they are from different species. In this work, the phylogeny of 42 slow-growing rhizobial strains, isolated from root nodules of cowpea, peanut, and mung bean from different geographical regions of China, was investigated using sequences from the 16S rRNA, atpD and glnII genes, and the 16S-23S rRNA intergenic spacer. The indigenous rhizobial strains from the cowpea miscellany could all be placed in the genus Bradyrhizobium , and Bradyrhizobium liaoningense and Bradyrhizobium yuanmingense were the main species. Phylogenies derived from housekeeping genes were consistent with phylogenies generated from the ribosomal gene. Mung bean rhizobia clustered only into B. liaoningense and B. yuanmingense and were phylogenetically less diverse than cowpea and peanut rhizobia. Geographical origin was significantly reflected in the phylogeny of mung bean rhizobia. Most cowpea rhizobia were more closely related to the 3 major groups B. liaoningense, B. yuanmingense, and Bradyrhizobium elkanii than to the minor groups Bradyrhizobium japonicum or Bradyrhizobium canariense . However, most peanut rhizobia were more closely related to the 2 major groups B. liaoningense and B. yuanmingense than to the minor group B. elkanii.     

Mycorrhizal fungi suppress aggressive agricultural weeds

Plant growth responses to arbuscular mycorrhizal fungi (AMF) are highly variable, ranging from mutualism in a wide range of plants, to antagonism in some non-mycorrhizal plant species and plants characteristic of disturbed environments. Many agricultural weeds are non mycorrhizal or originate from ruderal environments where AMF are rare or absent. This led us to hypothesize that AMF may suppress weed growth, a mycorrhizal attribute which has hardly been considered. We investigated the impact of AMF and AMF diversity (three versus one AMF taxon) on weed growth in experimental microcosms where a crop (sunflower) was grown together with six widespread weed species. The presence of AMF reduced total weed biomass with 47% in microcosms where weeds were grown together with sunflower and with 25% in microcosms where weeds were grown alone. The biomass of two out of six weed species was significantly reduced by AMF (?66% & ?59%) while the biomass of the four remaining weed species was only slightly reduced (?20% to ?37%). Sunflower productivity was not influenced by AMF or AMF diversity. However, sunflower benefitted from AMF via enhanced phosphorus nutrition. The results indicate that the stimulation of arbuscular mycorrhizal fungi in agro-ecosystems may suppress some aggressive weeds.     

Host-plant selectivity of rhizobacteria in a crop/weed model system

Belowground microorganisms are known to influence plants' performance by altering the soil environment. Plant pathogens such as cyanide-producing strains of the rhizobacterium Pseudomonas may show strong host-plant selectivity. We analyzed interactions between different host plants and Pseudomonas strains and tested if these can be linked to the cyanide sensitivity of host plants, the cyanide production of bacterial strains or the plant identity from which strains had been isolated. Eight strains (four cyanide producing) were isolated from roots of four weed species and then re-inoculated on the four weed and two additional crop species. Bacterial strain composition varied strongly among the four weed species. Although all six plant species showed different reductions in root growth when cyanide was artificially applied to seedlings, they were generally not negatively affected by inoculation with cyanide-producing bacterial strains. We found a highly significant plant species x bacterial strain interaction. Partitioning this interaction into contrasts showed that it was entirely due to a strongly negative effect of a bacterial strain (Pseudomonas kilonensis/brassicacearum, isolated from Galium mollugo) on Echinochloa crus-galli. This exotic weed may not have become adapted to the bacterial strain isolated from a native weed. Our findings suggest that host-specific rhizobacteria hold some promise as biological weed-control agents.     

Dynamics of weed infestation of main agricultural crops in Belarus

In Belarus about 300 weed plant species are marked, dominated by 30?–?40 species which are found on practically all the fields. Changes in the numbers of each weed species depend on many factors. The data of itinerary inspections of agricultural crops done in 1981?–?1985 and 1996?–?2003 show the specific composition change of the main dominating weed plants in comparison with 1939. The reasons promoting change of the specific composition of crop weed infestation are shown.     

The effects of gibberellin A3 on white clover during floral development

Hokkanen & Pimentel (1984) proposed a novel approach for the selection of biocontrol agents. They advocated the selection of agents from a relative of the weed plant rather than from the target weed species itself. The new relationship that such agents would have with the weed would be characterised by a relative lack of homeostasis compared with the old herbivore-plant associations traditionally used in weed biocontrol, and would consequently be more effective. There are few examples to support these contentions because of the traditional use of old herbivore-plant associations in weed biocontrol. In the present study, herbivore-plant associations in agricultural situations, which are analogous to agent-weed associations, are examined to assess the potential of new associations for weed biocontrol. The herbivores on 14 introduced crop plants which have salient similarities to the major weeds in the south-western Cape were surveyed: (a) 68% of the 188 insect and mite herbivores are indigenous species in new associations with these host plants, and (b) of the five most damaging pests on each of 13 of the crop plants, 53% are in new associations with the plants. Of the 40 most important agricultural pests in South Africa, 58% are in new associations, confirming these results. About 50% of the insect and mite herbivores in new associations with their host plants are oligophagous, indicating that new associations are not necessarily characterised by polyphagy and hence unsafe for use in weed biocontrol. We conclude that new associations between herbivore species and host plants have strong potential in weed biocontrol because (a) their frequency in agriculture indicates that they can easily be established in weed biocontrol situations, (b) they are as damaging as old associations and (c) they are not necessarily unsafe as regards host specificity. We therefore advocate the use of both the classical approach and that of Hokkanen & Pimentel (1984). Our support for Hokkanen & Pimentel (1984) is, however, based on evidence and rationale clearly different to theirs, and we provide novel guidelines, which can be routinely and practically applied in the selection of agents for weed biocontrol.     

Competition Experiments on Alien Weeds with Crops: Lessons for Measuring Plant Invasion Impact?

Can we quantify the impact of invasive species? Here we use the per-plant competitiveness of alien weeds on crops as a model of invasive species impact in general. We reviewed 97 weed–crop competition experiments in 32 papers that included 30 alien weed and 14 crop species. The majority (68.92%) were randomised block designs where the alien weed had been either added (additive experiments) or removed (removal experiments). We propose using the relative competition index to estimate the effect of alien species in all systems, specifying in each case the density and proportion of alien and native plants essayed. We found that the impact of the weed cannot be considered independently of the crop and, thus, we should be cautious in ranking weed species according to their competition effect. A similar situation can be postulated for alien plants interfering with native species. Invaded communities are not random assemblages, and researchers tend to study the most competitive alien plants. We also found that the effect of the weed on crop yield depends on the duration of the interference and the life-history stage of the weed–crop system at which the interaction takes place. We were not able to conduct a more rigorous comparative analysis of the impacts, such as a meta-analysis. To do this would require some measure of the variation of the competition effect such as standard deviation or standard error, which we found are almost never reported.     

Disturbance-mediated competition: the interacting roles of inundation regime and mechanical and herbicidal control in determining native and invasive plant abundance

Disturbance is a key component of many successful plant invasions. However, interactions among natural and anthropogenic disturbances and effects of these interacting disturbances on invasive plants and desired vegetation are rarely examined. We investigated the effect of anthropogenic disturbance (herbicidal and mechanical) along a natural inundation gradient (20–282 days) on the biomass and resource allocation of the invasive wetland plant, alligator weed (Alternanthera philoxeroides), and two co-occurring competitor plants, the introduced grass, kikuyu (Pennisetum clandestinum), and the native grass, couch (Cynodon dactylon), over a 2-year period. In the absence of additional disturbance, kikuyu biomass was negatively affected, alligator weed biomass was positively affected, and couch biomass was not affected by inundation disturbance. In addition, kikuyu was not affected by the selective removal of alligator weed, while couch increased in wetter habitats where kikuyu was absent due to inundation stress. This suggests that kikuyu is a superior competitor in drier habitats and inundation facilitates the invasion of alligator weed, while couch is an inferior competitor to both kikuyu and alligator weed and is therefore suppressed across its entire niche by these two introduced species. Mowing alone had no effect on the biomass of the species, suggesting the plants are equally tolerant of shoot removal. Selective herbicide reduced alligator weed biomass by 97.5% and the combination of selective herbicide and mowing reduced the biomass of alligator weed significantly more than herbicide alone, by 98.6% compared with un-manipulated controls. To predict community change and prevent sequential exotic plant invasions after weed removal, it is necessary to consider the interacting effects of disturbance and the niche space of invasive species in the local propagule pool.     

Weed vegetation of arable land in Central Europe: Gradients of diversity and species composition

Question: What are the main broad‐scale spatial and temporal gradients in species composition of arable weed communities and what are their underlying environmental variables? Location: Czech Republic and Slovakia. Methods: A selection of 2653 geographically stratified relevés sampled between 1954–2003 was analysed with direct and indirect ordination, regression analysis and analysis of beta diversity. Results: Major changes in weed species composition were associated with a complex gradient of increasing altitude and precipitation and decreasing temperature and base status of the soils. The proportion of hemicryptophytes increased, therophytes and alien species decreased, species richness increased and beta diversity decreased with increasing altitude. The second most important gradient of weed species composition was associated with seasonal changes, resulting in striking differences between weed communities developed in spring and summer. In summer, weed communities tended to have more neophytes, higher species richness and higher beta diversity. The third gradient reflected long‐term changes in weed vegetation over past decades. The proportion of hemicryptophytes and neophytes increased, while therophytes and archaeophytes decreased, as did species richness over time. The fourth gradient was due to crop plants. Cultures whose management involves less disturbances, such as cereals, harboured less geophytes and neophytes, and had higher species richness but lower beta diversity than frequently disturbed cultures, such as root crops. Conclusions: Species composition of Central European weed vegetation is mainly influenced by broad‐scale climatic and edaphic factors, but its variations due to seasonal dynamics and long‐term changes in agricultural management are also striking. Crop plants and crop‐specific management affect it to a lesser, but still significant extent.     

Weed genomics: new tools to understand weed biology

In spite of the large yield losses that weeds inflict on crops, we know little about the genomics of economically important weed species. Comparative genomics between plant model species and weeds, map-based approaches, genomic sequencing and functional genomics can play vital roles in understanding and dissecting weedy traits of agronomically important weed species that damage crops. Weed genomics research should increase our understanding of the evolution of herbicide resistance and of the basic genetics underlying traits that make weeds a successful group of plants. Here, we propose specific weed candidates as genomic models, including economically important plants that have evolved herbicide resistance on several occasions and weeds with good comparative genomic qualities that can be anchored to the genomics of Arabidopsis and Oryza sativa.     

Risk to non-target plants from Charidotis auroguttata (Chrysomelidae: Coleoptera), a potential biocontrol agent for cat’s claw creeper Macfadyena unguis-cati (Bignoniaceae) in Australia

The potential of the leaf beetle Charidotis auroguttata as a biocontrol agent for cat’s claw creeper Macfadyena unguis-cati (Bignoniaceae), an environmental weed in Australia, and risk to non-target plants was evaluated under quarantine conditions. In no-choice tests, C. auroguttata adults and larvae fed on many plant species across different families, but egg to adult development occurred only on the target weed. However, when neonate larvae from the target weed were transferred onto Myoporum boninense australe (Myoporaceae), a non-target native plant, 11.7% completed development, as compared to 95% of larvae that completed development on the target weed. Larval development on this non-target species also took twice as long as on the target weed. No larvae completed development on other test plants. In choice tests, leaf area consumption by adults and larvae was significantly more on the target weed than on other plants, and oviposition occurred only on the target weed. In the no-choice demography trials, adults laid eggs from the second week after emergence on the target weed, with an average of 0.286 eggs/female/day, resulting in an 18-fold increase in the adult population over 16 weeks. On My. boninense australe adult survival remained high, but oviposition commenced only from the 10th week after emergence with an average of 0.023 eggs/female/day, and none of the eggs developed into adults. In the choice demography trials, oviposition on the target weed was evident from the fourth week onwards, while on the non-target plant oviposition commenced only from the 14th week. Only 10% of total adults and 11.3% of total eggs were found on the non-target plant, and none of these eggs developed into adults. Although the biocontrol agent can ‘spill-over’ from the target weed to the non-target native plant and cause adult feeding damage, the non-target plant could not sustain a viable insect population on its own. This agent was not approved for field release in Australia due to perceived risk to non-target species.     

施肥和杂草多样性对土壤微生物群落的影响

常年使用化肥和除草剂以及农业新技术的高投入,使我国粮食主产区耕地出现了生产力降低、土壤生物多样性失调和污染严重等生态问题。采用磷脂脂肪酸(PLFA)方法来评估施肥和杂草多样性对冬小麦土壤微生物群落结构的影响。实验采用裂区实验设计,施肥作为主因素,杂草多样性作为次因素。化肥和有机肥两个施肥处理,在两个施肥处理中进行杂草多样性设置,实验盆中心种植作物(冬小麦8株),四周种植杂草(8株),杂草种类选择野燕麦、苜蓿、菊苣、播娘蒿。杂草多样性处理设为0、1、2、4种杂草处理,0种杂草处理仅种植作物,有6盆;1种杂草处理为每盆种1种杂草,有12盆;2种杂草处理为每盆种两种杂草,有12盆;4种杂草处理为每盆种4种杂草,有6盆。结果表明:在两种施肥处理中,增加杂草多样性显著增加了土壤碳氮比和p H值,碳氮比都是在4种杂草处理中最高。施化肥处理中,增加杂草多样性显著影响真菌和细菌比,真菌和细菌比在4种杂草处理中最大,显著高于0、1、2种杂草处理。在施有机肥处理中,增加杂草多样性显著影响阳性菌和阴性菌比,阳性菌和阴性菌比在0种杂草处理中最低,显著低于1、2、4种杂草处理。在两个施肥处理中,土壤碳氮比与各类群微生物量显著相关,杂草多样性通过改变土壤碳氮比改变微生物群落构成,并且微生物群落结构转变方式不同。     

The Ability of Aphis craccivora, A. gossypii and A. citricola to Transmit Single and Mixed Viruses to Cowpeas

The ability of populations of adult apterous Aphis craccivora, A. gossypii and A. citricola to transmit the cowpea aphid-borne virus (CAMV) and cucumber mosaic virus (CMV) to cowpea was investigated. CMV was more readily transmitted than CAMV by all three aphid species, but was most readily transmitted by A. craccivora and A. citricola, with transmissions ranging from 64–71 %. With CAMV, the infection level with A. gossypii was higher than with A. craccivora and both were more efficient than A. citricola, although the differences were not significant. With mixed infections of CAMV and CMV, there was a higher level of CAMV transmission using A. craccivora from sequential feeding when CMV-infected plants were fed on first followed by CAMV-infected plants, suggesting that cowpea plants were more prone to infection by CAMV when already infected by CMV.     

Weed and crop plants as reservoirs of peanut bud necrosis tospovirus and its occurrence in South India

Survey was carried out on occurrence of peanut bud necrosis disease (PBND) in Karnataka and Andhra Pradesh (AP) the major groundnut growing states in South India. PBND incidence was observed on crops, viz., greengram, blackgram, tomato, watermelon, cowpea, chilli, cucumber and sesame besides groundnut. The PBND incidence was higher in rainy season crops than in post-rainy season. Its proportion varied from place to place, and it was high in the vegetable and pulses growing areas. The weed plant species Achyranthus aspera, Acalypha indica, Alysicarpus rugosus, A. longifolia, Commelina bengalensis, Commelina jacobi, Corchorus trilocularis, Cyanoptis cuculetta, Eclipta alba, Euphorbia geniculata, Lochnera pusilla, Physalis minima and Sesbania rostrata were recorded for the first time as alternate hosts of PBND. Infection was also noticed for the first time on cucumber, muskmelon and sesame crops. Five weed species, viz., Achyranthus aspera, Ageratum conyzoides, Alysicarpus rugosus, Commelina bengalensis and Vigna trilobata were found abundant in AP and Karnataka. More infection was observed on Ageratum conyzoides (17.56%) compared with other weeds.     

Field host-specificity of the mile-a-minute weevil,Rhinoncomimus latipes Korotyaev (Coleoptera: Curculionidae)

The safe practice of biological control relies, in part, on an accurate evaluation of a potential agent’s host-specificity via testing through a “filter of safety”. The results of laboratory tests may differ from those obtained in open field host-specificity tests, where agents are able to use their full range of host-selection behaviors. It was hypothesized that Rhinoncomimus latipes (Coleoptera: Curculionidae), the biological control agent released against mile-a-minute weed, Persicaria perfoliata (Polygonaceae), would not feed or oviposit on nontarget plants in a two-phase, open field setting. Ten weevils were placed at the base of each of 13 test plant species in a randomized complete block design with six replicates. Weevils placed at the base of mile-a-minute weed were marked with yellow fluorescent dust, and yellow weevils were subsequently found only on mile-a-minute. Weevils placed at the base of nontarget plants (marked with red fluorescent dust) rapidly colonized mile-a-minute weed. Three hours after release, the number of R. latipes found on mile-a-minute weed was significantly higher than predicted by a random distribution of weevils on all test plants. The likelihood of finding more weevils on mile-a-minute compared to nontarget plant species was 31.0% at 3 h and increased to 96.5% at 44 h after release. Whereas prerelease studies showed feeding at low levels on 9 of the 13 plant species tested here, under open field conditions R. latipes did not feed on any nontarget plant species and dispersed from these plants. In an open field setting, where the weevil was able to use its full range of host-selection behaviors, there was no observed risk of nontarget effects for any species tested.