Effects of green fluorescent protein or beta-glucuronidase tagging on the accumulation and pathogenicity of a resistance-breaking Lettuce mosaic virus isolate in susceptible and resistant lettuce cultivars

The RNA genome of a resistance-breaking isolate of Lettuce mosaic virus (LMV-E) was engineered to express the jellyfish green fluorescent protein (GFP) or beta-glucuronidase (GUS) fused to the helper-component proteinase (HC-Pro) to study LMV invasion and spread in susceptible and resistant lettuce cultivars. Virus accumulation and movement were monitored by either histochemical GUS assays or detection of GFP fluorescence under UV light. The GFP- and GUS-tagged viruses spread systemically in the susceptible lettuce cultivars Trocadero and Vanguard, where they induced attenuated symptoms, compared with the wild-type virus. Accumulation of the GFP-tagged virus was reduced but less affected than in the case of the GUS-tagged virus. Systemic movement of both recombinant viruses was very severely affected in Vanguard 75, a lettuce cultivar nearly isogenic to Vanguard but carrying the resistance gene mo1(2). Accumulation of the recombinant viruses in systemically infected leaves was either undetectable (GUS-tag) or erratic, strongly delayed, and inhibited by as much as 90% (GFP-tag). As a consequence, and contrary to the parental virus, the recombinant viruses were not able to overcome the protection afforded by the mo1(2) gene. Taken together, these results indicate that GUS or GFP tagging of the HC-Pro of LMV has significant negative effects on the biology of the virus, abolishing its resistance-breaking properties and reducing its pathogenicity in susceptible cultivars.     

Lettuce mosaic virus: from pathogen diversity to host interactors

TAXONOMY: Lettuce mosaic virus (LMV) belongs to the genus Potyvirus (type species Potato virus Y) in the family Potyviridae. PHYSICAL PROPERTIES: The virion is filamentous, flexuous with a length of 750 nm and a width of 15 nm. The particles are made of a genomic RNA of 10 080 nucleotides, covalently linked to a viral-encoded protein (the VPg) at the 5' end and with a 3' poly A tail, and encapsidated in a single type of capsid protein. The molecular weight of the capsid protein subunit has been estimated electrophoretically to be 34 kDa and estimated from the amino acid sequence to be 31 kDa. GENOME ORGANIZATION: The genome is expressed as a polyprotein of 3255 amino-acid residues, processed by three virus-specific proteinases into ten mature proteins. HOSTS: LMV has a worldwide distribution and a relatively broad host range among several families. Weeds and ornamentals can act as local reservoirs for lettuce crops. In particular, many species within the family Asteraceae are susceptible to LMV, including cultivated and ornamental species such as common (Lactuca sativa), prickly (L. serriola) or wild (L. virosa) lettuce, endive/escarole (Cichorium endiva), safflower (Carthamus tinctorius), starthistle (Centaurea solstitialis), Cape daisy (Osteospermum spp.) and gazania (Gazania rigens). In addition, several species within the families Brassicaceae, Cucurbitaceae, Fabaceae, Solanaceae and Chenopodiaceae are natural or experimental hosts of LMV. Genetic control of resistance to LMV: The only resistance genes currently used to protect lettuce crops worldwide are the recessive genes mo1(1) and mo1(2) corresponding to mutant alleles of the gene encoding the translation initiation factor eIF4E in lettuce. It is believed that at least one intact copy of eIF4E must be present to ensure virus accumulation. TRANSMISSION: LMV is transmitted in a non-persistent manner by a high number of aphid species. Myzus persicae and Macrosiphum euphorbiae are particularly active in disseminating this virus in the fields. LMV is also seedborne in lettuce. The effectiveness of LMV transmission depends on the cultivar and the age of the seed carrier at the inoculation time. SYMPTOMS: The characteristic symptoms on susceptible lettuce cultivars are dwarfism, mosaic, distortion and yellowing of the leaves with sometimes a much reduced heart of lettuce (failure to form heads). The differences in virus strains, cultivars and the physiological stage of the host at the moment of the attack cause different symptom severity: from a very slight discoloration of the veins to severe necrosis leading to the death of the plant.     

A New Virulent Isolate of Clover Yellow Vein Virus on Phaseolus vulgaris in Bulgaria

Potyviruses are a common threat for snap bean production in Bulgaria. During virus surveys of bean plots in the south central region, we identified an isolate of Clover yellow vein virus (ClYVV), designated ClYVV 11B, by indirect ELISA and RT‐PCR causing severe mosaic symptoms and systemic necrosis. Indirect and direct ELISA using ClYVV antisera differentiated the ClYVV isolate from Bean yellow mosaic virus (BYMV), but serological analysis could not distinguish the Bulgarian isolate ClYVV 11B from an Italian ClYVV isolate used as a reference (ClYVV 505/7). RT‐PCR analyses with specific primers revealed that both isolates were ClYVV. Sequence analysis of an 800 bp fragment corresponding to the coat protein coding region showed 94% identity at the nucleotide level between the two isolates. Phylogenetic analyses of aligned nucleotide sequences available in the database confirmed the existence of two groups of isolates, but ClYVV 11B and ClYVV505/7 belonged to the same group. We compared the virulence of both isolates on a set of differential cultivars and 19 bean breeding lines resistant to Bean common mosaic virus (BCMV) and Bean common mosaic necrosis virus (BCMNV): Bulgarian isolate ClYVV 11B was able to infect systemically all tested bean differential cultivars and breeding lines including those with genotypes Ibc3 and Ibc2²; Italian isolate ClYVV 505/7 was not able to infect systemically some differentials with genotypes bc‐ubc1, bc‐ubc2², bc‐ubc2bc3, Ibc1², Ibc2², Ibc3. The role of bc3 gene as a source of resistance to potyviruses is discussed.     

Epidemiology of Pseudomonas cichorii,the Cause of Lettuce Midrib Rot

Bacterial midrib rot, caused by Pseudomonas cichorii, has become a serious threat to the production of greenhouse butterhead lettuce (Lactuca sativa L. var. capitata) in Belgium. Currently, there are no strategies for controlling this pathogen. Therefore, greenhouse experiments were conducted to obtain more knowledge about the epidemiology of P. cichorii on butterhead lettuce. Greenhouse butterhead lettuce becomes susceptible to lettuce midrib rot infections at head formation, and a single overhead irrigation with water containing 10² CFU/ml P. cichorii was sufficient to cause disease. The use of surface drip irrigation instead of overhead sprinkler irrigation significantly reduced midrib rot incidence in the greenhouse. P. cichorii isolates can be divided into subgroups based on BOX‐PCR genomic fingerprinting, with isolates belonging to subgroup C1 and C2 being more virulent than those of (or related to) subgroup C3. P. cichorii infections with distinct symptoms comparable to midrib rot have also been observed on field‐grown crisphead lettuce in California and Japan which, respectively, are referred to as ‘varnish spot’ or ‘tar’. We showed that symptom expression is strongly influenced by the lettuce cultivar group, irrespective of the P. cichorii isolate, resulting in varnish spot/tar on crisphead lettuce and midrib rot on butterhead or cutting group lettuce.     

The inheritance of resistance to Verticillium wilt caused by race 1 isolates of <Emphasis Type="Italic">Verticillium dahliae</Emphasis> in the lettuce cultivar La Brillante

Verticillium wilt of lettuce caused by Verticillium dahliae can cause severe economic damage to lettuce producers. Complete resistance to race 1 isolates is available in Lactuca sativa cultivar (cv.) La Brillante and understanding the genetic basis of this resistance will aid development of new resistant cultivars. F₁ and F₂ families from crosses between La Brillante and three iceberg cultivars as well as a recombinant inbred line population derived from L. sativa cv. Salinas 88 × La Brillante were evaluated for disease incidence and disease severity in replicated greenhouse and field experiments. One hundred and six molecular markers were used to generate a genetic map from Salinas 88 × La Brillante and for detection of quantitative trait loci. Segregation was consistent with a single dominant gene of major effect which we are naming Verticillium resistance 1 (Vr1). The gene described large portions of the phenotypic variance (R ² = 0.49–0.68) and was mapped to linkage group 9 coincident with an expressed sequence tag marker (QGD8I16.yg.ab1) that has sequence similarity with the Ve gene that confers resistance to V. dahliae race 1 in tomato. The simple inheritance of resistance indicates that breeding procedures designed for single genes will be applicable for developing resistant cultivars. QGD8I16.yg.ab1 is a good candidate for functional analysis and development of markers suitable for marker-assisted selection.     

Cytopathology of Anthers and Pollen From Lettuce Plants Infected by Lettuce Mosaic Virus

Thin sections of mature anthers and pollen grains from three lettuce (Lactuca sativa) plants infected with lettuce mosaic potyvirus (LMV) were studied by immunogold labelling. Labelled LMV particles were present externally on the exine of pollen grains from all plants, but were observed internally in the pollen grains from only one plant. Within mature pollen grains the virus particles were associated with the cytoplasmic bundle inclusions typical of infection by potyviruses. The tapetal plasmodium and the epidermal and endothecial layers of mature anthers from all infected plants also contained labelled virus particles, together with pinwheel and bundle inclusions.     

Response to potyvirus infection and genetic mapping of resistance loci to potyvirus infection in Lactuca

 We have investigated the interaction between two different potyviruses and resistant cultivars of Lactuca sativa. Turnip mosaic virus (TuMV) and lettuce mosaic virus (LMV) were used to inoculate several cultivars under different temperature regimes to characterize the resistance reaction. Resistance conferred by the recessive mo locus against LMV infection did not provide immunity. Virus accumulated in plant tissues to different levels depending on the genetic background of the cultivar, suggesting that several genes were involved in the resistance phenotype. Under temperature regimes that enhanced the hypersensitive reaction, resistant cultivars produced necrotic reactions. In contrast, resistance to TuMV infection conferred by the dominant Tu locus resulted in complete immunity in the plant. No virus accumulated in inoculated leaves nor was any necrotic reaction observed. The resistance loci were characterized at the genetic level by mapping them relative to molecular markers. Only weak linkages could be identified to mo, again supporting the hypothesis that several genes are involved. The Tu locus was mapped in two different crosses relative to several markers, the closest two linked at less than 1 cM. A high-resolution genetic map of the Tu locus was constructed by screening 500 F₂ individuals for recombinants around that locus. Received: 4 June 1996/Accepted: 15 November 1996     

Mutational analysis of plant cap-binding protein eIF4E reveals key amino acids involved in biochemical functions and potyvirus infection

The eukaryotic translation initiation factor 4E (eIF4E) (the cap-binding protein) is involved in natural resistance against several potyviruses in plants. In lettuce, the recessive resistance genes mo1¹ and mo1² against Lettuce mosaic virus (LMV) are alleles coding for forms of eIF4E unable, or less effective, to support virus accumulation. A recombinant LMV expressing the eIF4E of a susceptible lettuce variety from its genome was able to produce symptoms in mo1¹ or mo1² varieties. In order to identify the eIF4E amino acid residues necessary for viral infection, we constructed recombinant LMV expressing eIF4E with point mutations affecting various amino acids and compared the abilities of these eIF4E mutants to complement LMV infection in resistant plants. Three types of mutations were produced in order to affect different biochemical functions of eIF4E: cap binding, eIF4G binding, and putative interaction with other virus or host proteins. Several mutations severely reduced the ability of eIF4E to complement LMV accumulation in a resistant host and impeded essential eIF4E functions in yeast. However, the ability of eIF4E to bind a cap analogue or to fully interact with eIF4G appeared unlinked to LMV infection. In addition to providing a functional mutational map of a plant eIF4E, this suggests that the role of eIF4E in the LMV cycle might be distinct from its physiological function in cellular mRNA translation.     

Temporal and spatial spread of Lettuce mosaic virus in lettuce crops in central Spain: factors involved in Lettuce mosaic virus epidemics

Lettuce mosaic virus (LMV) is transmitted by aphid vectors in a nonpersistent manner as well as by seeds. The virus causes severe disease outbreaks in commercial lettuce crops in several regions of Spain. The temporal and spatial patterns of spread of LMV were studied in autumn 2002 in the central region of Spain. Symptomatic lettuce (var. Cazorla) plant samples were collected weekly, first at the seedling stage from the greenhouse nursery and later outdoors after transplantation. The exact position of symptomatic plants sampled in the field was recorded and then material was tested by enzyme‐linked immunosorbent assay to assess virus infection. Cumulative spatial data for infected plants at different growth stages were analysed using spatial analysis by distance indices. For temporal analysis, the monomolecular, Gompertz, logistic and exponential models were evaluated for goodness of fit to the entire set of disease progress data obtained. The results indicated that the disease progress curve of LMV epidemics in the selected area is best described by a Gompertz model and that the epidemic follows a polycyclic disease progression. Our data suggest that secondary cycle of spread occurs when noncolonising aphid species land on the primary infected plants (probably coming from infected seed) and move to adjacent plants before leaving the crop. The role of weeds growing close to lettuce fields as potential inoculum sources of virus and the aphid species most likely involved in the transmission of LMV were also identified.     

The incidence and distribution of viruses infecting lettuce, cultivated Brassica and associated natural vegetation in Spain

A virus survey was conducted during the spring and autumn of 2001 and 2002 to determine the presence, prevalence and distribution in Spain of the viruses that are most commonly found infecting lettuce and Brassica worldwide. Crop plants showing virus symptoms from the principal lettuce and Brassica-growing regions of Spain, and some samples of the annual and perennial flora nearby, were tested by enzyme-linked immunosorbent assays using specific commercial antibodies against the following viruses: Alfalfa mosaic virus (AMV), Broad bean wilt virus 1 (BBWV-1), Beet western yellows virus (BWYV), Cauliflower mosaic virus (CaMV), Cucumber mosaic virus (CMV), Lettuce mosaic virus (LMV), Pea seed-borne mosaic virus (PSbMV), Turnip mosaic virus (TuMV) and Tomato spotted wilt virus (TSWV). Samples were also tested with a Potyvirus genus antibody. Virus incidence was much lower in spring than in autumn, especially in 2001. In spring 2002, CMV and LMV were the most prevalent viruses in lettuce, while CaMV was the most important virus present in Brassica crops grown in Navarra, followed by CMV and BWYV. In the autumn, the spectrum of viruses was different; potyviruses were widespread in lettuce grown in Madrid, but TSWV and BWYV were predominant in the Murcia region. The prevalent Potyvirus detected in lettuce fields was LMV, but none of the samples collected were positive for PSbMV or TuMV. In Brassica crops, TSWV was the most abundant in autumn-sown crops, especially in the Navarra region. All of the viruses present in lettuce and Brassica were also frequently detected in their associated natural vegetation at the same time, suggesting that they probably play an important role as virus reservoirs. Sonchus spp. were particularly common and were frequently infected with CMV, LMV and BWYV. Another common species, Chenopodium album, was often infected with TSWV and BWYV. Multiple infections were common, especially in non-crop plants, and the most common combination was BWYV and TSWV. The role of weeds in the epidemiology of viruses that infect lettuce and Brassica crops in Spain is discussed.     

The eukaryotic translation initiation factor 4E controls lettuce susceptibility to the Potyvirus Lettuce mosaic virus

The eIF4E and eIF(iso)4E cDNAs from several genotypes of lettuce (Lactuca sativa) that are susceptible, tolerant, or resistant to infection by Lettuce mosaic virus (LMV; genus Potyvirus) were cloned and sequenced. Although Ls-eIF(iso)4E was monomorphic in sequence, three types of Ls-eIF4E differed by point sequence variations, and a short in-frame deletion in one of them. The amino acid variations specific to Ls-eIF4E(1) and Ls-eIF4E(2) were predicted to be located near the cap recognition pocket in a homology-based tridimensional protein model. In 19 lettuce genotypes, including two near-isogenic pairs, there was a strict correlation between these three allelic types and the presence or absence of the recessive LMV resistance genes mo1(1) and mo1(2). Ls-eIF4E(1) and mo1(1) cosegregated in the progeny of two separate crosses between susceptible genotypes and an mo1(1) genotype. Finally, transient ectopic expression of Ls-eIF4E restored systemic accumulation of a green fluorescent protein-tagged LMV in LMV-resistant mo1(2) plants and a recombinant LMV expressing Ls-eIF4E degrees from its genome, but not Ls-eIF4E(1) or Ls-eIF(iso)4E, accumulated and produced symptoms in mo1(1) or mo1(2) genotypes. Therefore, sequence correlation, tight genetic linkage, and functional complementation strongly suggest that eIF4E plays a role in the LMV cycle in lettuce and that mo1(1) and mo1(2) are alleles coding for forms of eIF4E unable or less effective to fulfill this role. More generally, the isoforms of eIF4E appear to be host factors involved in the cycle of potyviruses in plants, probably through a general mechanism yet to be clarified.     

A Novel Satellite RNA of Cucumber Mosaic Virus Induces Unique Line-pattern Mosaic Symptoms in Tobacco

The CMV(YW) isolate of cucumber mosaic virus (CMV) induced unique line‐pattern mosaic symptoms in systemically infected leaves of tobacco (Nicotiana tabacum cv. Ky57). By northern hybridization analysis using cDNA to CMV(Y) satellite RNA as a probe, it was confirmed that CMV(YW) contained a satellite RNA. which was designated sat‐YW RNA; this was 388 nucleotides in length and did not have either a conserved domain that induces necrosis in tomato or chlorosis in tobacco. CMV(YW) free of sat‐YW RNA. which was isolated by the single lesion isolation method using Chenopodium amaranticolor, did not induce the unique line‐pattern mosaic symptom. Furthermore, the sat‐YW RNA‐mediated line‐pattern mosaic symptom was also induced by in vitro transcribed infectious sat‐YW RNA in tobaccos infected with either CMV(YW) or CMV(Y) genomic RNA. These results clearly demonstrated that sat‐YW RNA induces the unique line‐pattern mosaic symptom on CMV‐infected tobaccos.     

An assessment of genetic variability in somacloned lettuce plants (Lactuca sativa) and their offspring

Plants were regenerated from callus derived from cotyledons and first true-leaves of the lettuce cultivars Salad Bowl, Lobjoits Cos and Pennlake. Sexual progeny of these regenerants were assessed under glasshouse and field conditions for variation including reaction to lettuce mosaic virus (LMV) and downy mildew (Bremia lactucae). All three cultivars exhibited somaclonal variation. Mutations detected at the seedling stage included reduced vigour, albinism and changes in chlorophyll content, with most being recessive. Variation for leaf shape and vigour was detected in mature plants. One line exhibited increased yield and chlorophyll content together with early flowering. Enhanced and reduced susceptibility to both LMV and B. lactucae were observed. Reduced susceptibility to B. lactucae was indicated by an extended latent period following inoculation in two lines. Reduced susceptibility to LMV in glasshouse trials could not be confirmed in the field although one such line exhibited an improved yield and a second line segregated 1:1 in glasshouse tests for plants which were obviously infected and those without symptoms. All variable lines were diploid.     

Lettuce mosaic virus pathogenicity determinants in susceptible and tolerant lettuce cultivars map to different regions of the viral genome

Full-length infectious cDNA clones were constructed for two isolates (LMV-0 and LMV-E) of Lettuce mosaic virus (LMV), a member of the genus Potyvirus. These two isolates differ in pathogenicity in susceptible and tolerant-resistant lettuce cultivars. In susceptible plants, LMV-0 induces mild mosaic symptoms, whereas LMV-E induces severe stunting, leaf deformation, and a necrotic mosaic. In plants carrying either of the two probably allelic recessive resistance genes mol1 or mol2, LMV-0 is restricted partially to the inoculated leaves. When a systemic invasion does occur, however, symptoms fail to develop. LMV-E overcomes the protection afforded by the resistance genes, resulting in systemic mosaic symptoms. Analysis of the behavior of recombinants constructed between the two virus isolates determined that the HC-Pro protein of LMV-E causes the severe stunting and necrotic mosaic induced by this isolate in susceptible cultivars. In contrast, the ability to overcome mol resistance and induce symptoms in the resistant-tolerant cultivars was mapped to the 3' half of the LMV-E genome. These results indicate that the ability to induce severe symptoms and to overcome the protection afforded by the recessive genes mol1 or mol2 are independent phenomena.     

Tolerance to ryegrass mosaic virus and its inheritance

Plant yield within and between four cultivars of perennial ryegrass infected with ryegrass mosaic virus (RMV) was closely related to symptom severity. Distribution of symptom severity was continuous in four perennial ryegrass and four Italian ryegrass cultivars infected with a severe RMV isolate, and also in another perennial ryegrass cultivar infected with a severe isolate of the virus, a mild one and one of intermediate severity. Symptom expression was polygenically inherited in both Italian (cv. RvP) and perennial (cv. S.24) ryegrass. Both additive and non-additive genetic variation was present in RvP, but the variation in S.24 was additive only. No significant maternal inheritance was present in either species.     

Studies on plant growth-regulating substances LV. The Plant growth-promoting properties of some β-aryl-propionic acids

Severe diseases of pepper (Capsicum annuum), tomato (Lycopersicon esculentum), eggplant (Solanum melongena) and tomato eggplant (Solanum integrifolium) in West Africa were induced by pepper veinal mottle virus (PVMV). Five selected virus isolates were serologically similar and readily transmissible by aphids in the non-persistent manner, but they differed in host range and/or symptoms induced in some susceptible species. One isolate from eggplant failed to infect pepper, Chenopodium quinoa and C. amaranticolor, and induced only local infections in tomato. An isolate from tomato failed to infect eggplant, and an isolate from tomato eggplant induced severe stunting in Physalis floridana. The type strain, like the isolate from tomato, failed to infect Nicotiana tabacum systemically, but each caused severe systemic leaf and stem necrosis in tomato. None of the isolates infected S. melongena cv. Long Purple, suggesting that PVMV might be controlled in this and perhaps other crop species by the use of immune or tolerant cultivars. All five isolates were serologically related to potato virus Y and some to six of 12 other potyviruses.     

Differences in the Induction of Defence Responses in Resistant and Susceptible Muskmelon Plants Infected with Colletotrichum lagenarium

Defence reactions occurring in resistant (cv. Gankezaomi) and susceptible (cv. Ganmibao) muskmelon leaves were investigated after inoculating with Colletotrichum lagenarium. Lesion restriction in resistant cultivars was associated with the accumulation of hydrogen peroxide (H₂O₂). The activity of antioxidants catalase (CAT) and peroxidase (POD) significantly increased in both cultivars after inoculation, while levels of both CAT and POD activity were significantly higher in the resistant cultivar. Ascorbate peroxidase (APX) increased in both cultivars after inoculation, and level of APX activity was significantly higher in the resistant cultivar. Glutathione reductase (GR) activity significantly increased in both cultivars following inoculation, but was higher in the resistant cultivar, resulting in higher levels of ascorbic acid (AsA) and reduced glutathione (GSH). Phenylalanine ammonia lyase (PAL) significantly increased in inoculated leaves of both cultivars, resulting in higher levels of total phenolic compounds and flavonoids. The pathogenesis‐related proteins chitinase (CHT) and β‐1, 3‐glucanase (GLU) significantly increased following inoculation with higher activity in the resistant cultivar. These findings show that resistance of muskmelon plants against C. lagenarium is associated with the rapid accumulation of H₂O₂, resulting in altered cellular redox status, accumulation of pathogenesis‐related proteins, activation of phenylpropanoid pathway to accumulation of phenolic compounds and flavonoids.     

The susceptibility of barley cultivars to barley yellow mosaic virus (BaYMV) and its fungal vector, Polymyxa graminis

In glasshouse experiments, barley seedlings were inoculated with barley yellow mosaic virus (BaYMV) either mechanically or by using zoospores or cystosori of a viruliferous isolate of the vector, Polymyxa graminis, maintained on barley in sand culture. Experiments using mechanical inoculation showed that seedlings became more resistant with age. Consistent cultivar differences were obtained: cvs Maris Otter and Halcyon were very susceptible and cv. Athene seemed immune. Symptoms developed more rapidly at 23 than at 17 or 11 ^oC. After vector inoculation, symptoms developed more slowly than after mechanical inoculation but cultivar ranking was similar. Cultivars did not differ in susceptibility to the vector, as measured by zoospore production on their roots. Spring barley cultivars supported the growth of the vector which remained viruliferous and some showed symptoms although, in the field, symptoms do not appear on spring-sown crops.     

Mix‐planting pubescent and glabrous cassava affects abundance of Typhlodromalus aripo and its prey mite Mononychellus tanajoa

There is an increasing awareness that vegetation diversity can affect herbivore and natural enemy abundance and that plants can play a major role in directly manipulating natural enemy abundance for protection against herbivore attacks. Using data from cassava fields, we aimed at (i) testing the capacity of the predatory mite Typhlodromalus aripo to control the herbivorous mite Mononychellus tanajoa in a chemical exclusion trial; and (ii) testing, based on the differential preference by T. aripo for cassava cultivars, how combinations of two morphologically different cassava cultivars with differential suitability to the predator can improve its population densities on the non‐favourable cultivar, thereby reducing M. tanajoa densities with subsequent increases in cassava yield. The study was conducted in a cassava field in Benin, West Africa. The experiments confirmed that T. aripo effectively suppresses M. tanajoa populations on both cultivars and showed, in the no‐predator‐exclusion experiments, that cultivar combinations have significant effects on M. tanajoa and T. aripo densities. Indeed, T. aripo load on the non‐preferred cultivar was lowest in subplots where the proportion of T. aripo‐preferred cultivar was also low, while, and as expected, M. tanajoa load on the non‐preferred cultivar showed decreasing trends with increasing T. aripo densities. The possible mechanisms by which cultivar mixing could increase predator load on the non‐favourable cultivar were discussed. Our data showed that appropriate cultivar combinations effectively compensate for morphologically related differences in natural enemy abundance on a normally predator‐deficient cultivar, resulting in lower pest densities on the non‐favourable cultivar. In practical terms, this strategy could, in part, enhance adoption of cultivars that do not support sufficient levels of natural enemies for pest control.     

Re-evaluation of the effects of growth regulators on callus induction and shoot regeneration in <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of lettuce

Lettuce (Lactuca sativa) transformation varies by genotype. Various culture parameters have been studied in order to improve the transformation efficiency of lettuce cultivars. However, no improved transformation procedure for recalcitrant lettuce cultivars has yet been established. Here, we demonstrate the effects of varying concentrations and distinct combinations of growth regulators on recalcitrant lettuce transformation efficiency. More precisely, we assessed differences in the effects of several growth regulator combinations, including N-6(2-isopentenyl)-adenine (2ip), on induction of callus and regeneration of shoots after co-cultivation with Agrobacterium. When two commercial recalcitrant cultivars, Red Romaine and Bibb, were cultured on a medium with 2ip 1 mg l⁻¹, IAA 0.1 mg l⁻¹, and subsequently transferred to a second medium with BA 0.4 mg l⁻¹, NAA 0.05 mg l⁻¹ for selection and shoot regeneration, transformation efficiencies reached 8 and 9%, respectively. Stable integration and transmission of the transgene in T1 generation plants were confirmed by molecular analysis. This procedure represents a simple, efficient, and general means of transforming various lettuce cultivars, including recalcitrant commercial cultivars.