Aphid Honeydew Quality as a Food Source for Parasitoids Is Maintained in Bt Cotton

Bt-transgenic cotton has proven to be highly efficient in controlling key lepidopteran pests. One concern with the deployment of Bt cotton varieties is the potential proliferation of non-target pests. We previously showed that Bt cotton contained lower concentrations of insecticidal terpenoids as a result of reduced caterpillar damage, which benefited the aphid Aphis gossypii. It is thus important that non-target herbivores are under biological control in Bt cotton fields. The induction or lack of induction of terpenoids could also influence the quality of aphid honeydew, an important food source for beneficial insects. We therefore screened A. gossypii honeydew for cotton terpenoids, that are induced by caterpillars but not the aphids. We then tested the influence of induced insect-resistance of cotton on honeydew nutritional quality for the aphid parasitoid Lysiphlebus testaceipes and the whitefly parasitoid Eretmocerus eremicus. We detected the cotton terpenoids gossypol and hemigossypolone in A. gossypii honeydew. Although a feeding assay demonstrated that gossypol reduced the longevity of both parasitoid species in a non-linear, dose-dependent manner, the honeydew was capable of sustaining parasitoid longevity and reproduction. The level of caterpillar damage to Bt and non-Bt cotton had no impact on the quality of honeydew for the parasitoids.These results indicate that the nutritional quality of honeydew is maintained in Bt cotton and is not influenced by induced insect resistance.     

Effects of transgenic Bt cotton on overwintering characteristics and survival of Helicoverpa armigera

The effects of transgenic Bt cotton on the overwintering generation of the cotton bollworm, Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae), are unknown. We hypothesized that a Bt cotton diet may adversely affect fitness of this generation and examined fresh weight, lipids, glycogens, low-molecular-weight sugars and SCPs (supercooling points) of pupae, as well as survival of larvae, diapausing pupae and adult emergence in comparison with controls. Field and laboratory experiments showed that larvae fed on Bt cotton had a decreased pupation rate, and fewer entered diapause and emerged as adults compared with larvae fed non-Bt cotton. Furthermore, larvae fed Bt cotton had reduced pupal weight, glycogen content and trehalose levels both in diapausing and in non-diapausing pupae, and only diapausing pupae had an increased SCP compared to controls. The SCPs of diapausing pupae reared on Bt cotton were significantly higher than those reared on non-Bt cotton. The trehalose levels of diapausing pupae reared on Bt cotton were significantly lower than those of larvae reared on non-Bt cotton. Thus, these results suggest that a Bt cotton diet weakens the preparedness of cotton bollworm for overwintering and reduces survival of the overwintering generation, which will in turn reduce the density of the first generation in the following year. Effects of transgenic Bt cotton on the overwintering generation of cotton bollworm appear to have significantly contributed to the suppression of cotton bollworm observed throughout northern China in the past decade.     

Field Evaluation of Bt Cotton Crop Impact on Nontarget Pests: Cotton Aphid and Boll Weevil

Bt cotton plants expressing Cry1Ac protein have high specificity for the control of lepidopteran larvae. However, studies conducted in several countries have shown these plants have a differential impact on nontarget herbivores. The aim of this study was to compare the colonization rates and population abundance of the cotton aphid, Aphis gossypii Glover (Hemiptera: Aphididae) and the boll weevil, Anthonomus grandis Boheman (Coleoptera: Curculionidae), in plots of Bt (Nuopal) and non-Bt cotton (Delta Opal) in an experimental field in Brasilia, DF, Brazil. No difference was observed in the preference and colonization by winged aphids to plants from the two treatments. There was no significant difference in abundance of wingless aphids or in the production of winged aphids between treatments. Apparently, the parameters that control factors such as fecundity, survival, and dispersal were similar on both Bt and non-Bt plants. Monitoring of plants for coccinellids, a specialist predator of aphids, and ants that act on the dispersal of aphids among plants showed no significant difference between Bt and non-Bt plants, supporting the inference above. Regarding the effect on boll weevil, there was also no significant difference between treatments in the total number of fruiting structures attacked in each plot, the percentage of fruiting structures attacked per plant or on the number of weevils emerging from fruits with boll weevil damage from egg-laying, when damaged fruit samples were held in the laboratory. Based on these results, we conclude that there is no impact of Bt cotton crop expressing Cry1Ac on the nontarget herbivores tested under field conditions.     

Phage loss and the breakdown of a defensive symbiosis in aphids

Terrestrial arthropods are often infected with heritable bacterial symbionts, which may themselves be infected by bacteriophages. However, what role, if any, bacteriophages play in the regulation and maintenance of insect–bacteria symbioses is largely unknown. Infection of the aphid Acyrthosiphon pisum by the bacterial symbiont Hamiltonella defensa confers protection against parasitoid wasps, but only when H. defensa is itself infected by the phage A. pisum secondary endosymbiont (APSE). Here, we use a controlled genetic background and correlation-based assays to show that loss of APSE is associated with up to sevenfold increases in the intra-aphid abundance of H. defensa. APSE loss is also associated with severe deleterious effects on aphid fitness: aphids infected with H. defensa lacking APSE have a significantly delayed onset of reproduction, lower weight at adulthood and half as many total offspring as aphids infected with phage-harbouring H. defensa, indicating that phage loss can rapidly lead to the breakdown of the defensive symbiosis. Our results overall indicate that bacteriophages play critical roles in both aphid defence and the maintenance of heritable symbiosis.     

Effects of the polydnavirus of Cotesia congregata on the immune system and development of non-habitual hosts of the parasitoid

Polydnaviruses (PDV) are obligate mutualistic symbionts found in association with some groups of parasitic Hymenoptera. In these groups, they suppress the immune response of the parasitoid’s host and are required for successful parasitoid reproduction. Several PDV effects have been described in different experimental systems, but no clear picture of PDV mode of immunosuppression has emerged. No study to date has directly tested if PDV modes of action are evolutionarily conserved or divergent among parasitoid taxa within the Ichneumonoidea. We hypothesize the divergence in PDV mode of immunosuppression can be detected by identifying points of divergence in the immune response of different host species to PDV from one parasitoid species. This study tests the effects of purified PDV from Cotesia congregata on the immune response of three larval lepidopteran species that naturally are hosts of parasitoid species that differ in taxonomic relatedness to C. congregata. Here we demonstrate that despite associations with distantly related parasitoids (Ichneumonidae and Braconidae), Manduca sexta and Heliothis virescens showed similar patterns of increased glucose dehydrogenase (GLD) activity, suppressed cellular encapsulation in vitro, and increased time to pupation. In contrast, Lymantria dispar showed no response to C. congregata PDV across any of the parameters measured, even though it has an evolutionary association with several parasitoids closely related to C. congregata and within the Microgastrinae. The PDV immunosuppression in H. virescens and M. sexta does not correlate with host molecular phylogeny either. The suborganismal effects shown in M. sexta and H. virescens translated into significantly reduced pupation success in M. sexta only. Results demonstrate that while some PDV modes of immunosuppression in hosts may be divergent, others may be conserved across broad host groups.     

Electroantennogram (EAG) responses of Microplitis croceipes and Cotesia marginiventris and their lepidopteran hosts to a wide array of odor stimuli: Correlation between EAG response and degree of host specificity?

In order to test whether the electroantennogram (EAG) response spectrum of an insect correlates to its degree of host specificity, we recorded EAG responses of two parasitoid species with different degrees of host specificity, Microplitis croceipes (specialist) and Cotesia marginiventris (generalist), to a wide array of odor stimuli including compounds representing green leaf volatiles (GLVs), herbivore-induced plant volatiles (HIPV), ecologically irrelevant (not used by the parasitoid species and their hosts for host location) plant volatiles, and host-specific odor stimuli (host sex pheromones, and extracts of host caterpillar body and frass). We also tested the EAG responses of female moths of the caterpillar hosts of the parasitoids, Heliothis virescens and Spodoptera exigua, to some of the odor stimuli. We hypothesized that the specialist parasitoid will have a narrower EAG response spectrum than the generalist, and that the two lepidopteran species, which are similar in their host plant use, will show similar EAG response spectra to plant volatiles. As predicted, the specialist parasitoid showed greater EAG responses than the generalist to host-specific odor and one HIPV (cis-3-hexenyl butyrate), whereas the generalist showed relatively greater EAG responses to the GLVs and unrelated plant volatiles. We detected no differences in the EAG responses of H. virescens and S. exigua to any of the tested odor.     

Host selection and parasitism behavior of Lysiphlebus testaceipes: Role of plant,aphid species and instar

The aphid parasitoid Lysiphlebus testaceipes is a potentially valuable biological control agent of Aphis gossypii a major worldwide pest of cotton. One means of increasing the abundance of a biological control agent is to provide an alternative host habitat adjacent to cropping, from which they can provide pest control services in the crop. Host selection and parasitism rate of an alternative host aphid, Aphis craccivora by L. testaceipes were studied in a series of experiments that tested its host suitability relative to A. gossypii on cotton, hibiscus and mungbean. Host acceptance, as measured by number of ovipositions was much greater in A. craccivora compared to A. gossypii, while more host aphids were accepted on mungbean than cotton. When given a choice L. testaceipes attacks more 4th instar and adult stages (63% and 70%, respectively) of both hosts than 2nd instar nymphs (47%). In a switching (host choice) experiment, L. testaceipes preferentially attacked A. craccivora on mungbean over A. gossypii on cotton. Observations of parasitoid contact with A. gossypii cornicle secretion suggest it provides a useful deterrent against parasitoid attack. From these experiments it appears L. testaceipes has a preference for A. craccivora and mungbean compared to A. gossypii and cotton, in this respect using A. craccivora and mungbean as alternative habitat may not work as the parasitoid is unlikely to switch away from its preferred host.     

Evaluation of four aphidiine parasitoids for biological control ofAphis gossypii

Four aphidiine parasitoid species (Hymenoptera: Braconidae) were evaluated with respect to their potential to controlAphis gossypii Glover (Homoptera: Aphididae) in glasshouse cucumbers. In a laboratory experiment thirty cotton aphids were offered to individual females for two hours.Aphidius matricariae Haliday parasitized less than six percent of the aphids and was ruled out as potential biological control agent.Ephedrus cerasicola Stary andLysiphlebus testaceipes Cresson parasitized 23 and 26 percent of the aphids, respectively.Aphidius colemani Viereck parasitized 72 to 80 percent of the aphids. With the latter three species, experiments were performed in small glasshouses with cucumbers (Cucumis sativus L. cv. ‘Aramon’). As in the laboratory testA. colemani performed best; significantly more colonies were found and parasitization rates in the colonies were higher byA. colemani than byE. cerasicola andL. testaceipes. Because of the good correspondence between laboratory and glasshouse experiments, it is suggested that bad performance of an aphid parasitoid species in a simple laboratory trial might be sufficient evidence to disregard this species for further tests.     

Effect of Bt genetic engineering on indirect defense in cotton via a tritrophic interaction

We present a tritrophic analysis of the potential non-intended pleiotropic effects of cry1Ac gene derived from Bacillus thurigiensis (Bt) insertion in cotton (DeltaPine 404 Bt Bollgard® variety) on the emission of herbivore induced volatile compounds and on the attraction of the egg parasitoid Trichogramma pretisoum (Hymenoptera: Trichogrammatidae). Both the herbivore damaged Bt variety and its non-Bt isoline (DeltaPine DP4049 variety) produced volatiles in higher quantity when compared to undamaged plants and significantly attracted the egg parasitoids (T. pretiosum) when compared to undamaged plants. However, Trichogramma pretiosum did not differentiate between the transgenic and nontransgenic varieties, suggesting that the ratios between the compounds released by herbivory damaged -Bt cotton and herbivory damaged-nonBt cotton did not change significantly. Finally, no detrimental effect of the Bt genetic engineering was detected related to the volatile compounds released by Bollgard cotton on the behavior of the natural enemy studied.     

Leaf surface factors of transgenic Bt cotton associated with the feeding behaviors of cotton aphids: A case study on non-target effects

The present paper reports case study results of the risk assessment of transgenic Bt cotton on a non-target pest, cotton aphid, Aphis gossypii. Several types of techniques, i.e., electrical penetration graph (EPG), light and electron microscopy, bioassays and chemical analysis, were applied to investigate physical and chemical leaf factors of 2 transgenic Bt cotton lines (GK12 and GK19) and their pa-rental non-Bt cotton line (Simian3) associated with searching and feeding behaviors of cotton aphids on leaves or leaf extracts of cotton plants. EPG results showed that there were some differences among behaviors of cotton aphids on 2 Bt cotton and 1 non-Bt cotton lines. Cotton aphids performed similarly to leaf surface extracts from 3 cotton lines; and leaf surface chemicals, mainly volatiles and waxes, were almost identical in the components and concentrations among the cotton lines. However, three cotton lines were quite different from each other in the densities of certain kinds of covering trichomes. Therefore, the relationships between the physical characteristics and the searching behaviors of cotton aphids on the three cotton lines were constructed as the regression equations. Glandular trichomes and covering trichomes with 5 branches influenced the cotton aphids' searching behaviors effectively; and other trichomes with other branches affected aphids in varying ways. These results demonstrated that leaf surface physical factors of transgenic Bt cotton lines different from their parental non-Bt line could affect the penetration behaviors of non-target cotton aphids. Cotton aphids penetrate and feed more easily on two Bt cotton lines than on the non-Bt cotton line.     

The role of herbivore‐ and plant‐related experiences in intraspecific host preference of a relatively specialized parasitoid

Parasitoids use odor cues from infested plants and herbivore hosts to locate their hosts. Specialist parasitoids of generalist herbivores are predicted to rely more on herbivorederived cues than plant-derived cues. Microplitis croceipes (Cresson)(Hymenoptera: Braconidae) is a relatively specialized larval endoparasitoid of Heliothis virescens (F.)(Lepidoptera: Noctuidae), which is a generalist herbivore on several crops including cotton and soybean. Using M. croceipes/H. virescens as a model system, we tested the following predictions about specialist parasitoids of generalist herbivores:(i) naive parasitoids will show innate responses to herbivore-emitted kairomones, regardless of host plant identity and (ii) herbivore-related experience will have a greater influence on intraspecific oviposition preference than plant-related experience. Inexperienced (naive) female M. croceipes did not discriminate between cotton-fed and soybean-fed H. virescens in oviposition choice tests, supporting our first prediction. Oviposition experience alone with either host group influenced subsequent oviposition preference while experience with infested plants alone did not elicit preference in M. croceipes, supporting our second prediction. Furthermore, associative learning of oviposition with host-damaged plants facilitated host location. I terestingly, naive parasitoids attacked more soybeathan cotton-fed host larvae in two-choice tests when a background of host-infested cotton odor was supplied, and vice versa. This suggests that plant volatiles may have created an olfactory contrast effect. We discussed ecological significance of the results and concluded that both plant- and herbivore-related experiences play important role in parasitoid host foraging.     

Extended in vitro culture of Microplitis croceipes teratocytes and secretion of TSP14 protein

Teratocytes, cells which originate from the serosal membrane of some Braconidae and Scelionidae, can be found in the hemocoel of permissive hosts during part or all of the developmental time of the parasitoid larva. Teratocytes from Microplitis croceipes are known to secrete biologically active proteins, which contribute to developmental arrest and failure to pupate of Heliothis virescens larvae. One such protein, which has a molecular weight of approximately 14 kDa is called TSP14. The presence of parasitoid larvae is essential to maintain teratocytes under in vitro conditions with protein-free EX-CELL 400. The teratocyte viability was maintained in vitro for at least 12 days in the presence of larvae when medium was exchanged every three days. Western blots show that TSP14 was secreted during the entire period of exchanges. In the absence of parasitoid larvae, teratocyte viability was only 30% by day 6 and no TSP14 had been secreted. In the absence of parasitoid larvae, teratocytes maintained in vitro in EX-CELL 400 medium supplemented with 10% FBS remained viable for at least nine days and secreted TSP14 for at least six days. This suggests that parasitoid larval secretions are sufficient but not uniquely essential to maintain teratocyte viability. Parasitoid larvae maintained in the absence of teratocytes did not secrete TSP14 and their secretory products did not inhibit pupation of H. virescens larvae.     

Bt crop effects on functional guilds of non-target arthropods: a meta-analysis

Background

Uncertainty persists over the environmental effects of genetically-engineered crops that produce the insecticidal Cry proteins of Bacillus thuringiensis (Bt). We performed meta-analyses on a modified public database to synthesize current knowledge about the effects of Bt cotton, maize and potato on the abundance and interactions of arthropod non-target functional guilds.

Methodology/Principal Findings

We compared the abundance of predators, parasitoids, omnivores, detritivores and herbivores under scenarios in which neither, only the non-Bt crops, or both Bt and non-Bt crops received insecticide treatments. Predators were less abundant in Bt cotton compared to unsprayed non-Bt controls. As expected, fewer specialist parasitoids of the target pest occurred in Bt maize fields compared to unsprayed non-Bt controls, but no significant reduction was detected for other parasitoids. Numbers of predators and herbivores were higher in Bt crops compared to sprayed non-Bt controls, and type of insecticide influenced the magnitude of the difference. Omnivores and detritivores were more abundant in insecticide-treated controls and for the latter guild this was associated with reductions of their predators in sprayed non-Bt maize. No differences in abundance were found when both Bt and non-Bt crops were sprayed. Predator-to-prey ratios were unchanged by either Bt crops or the use of insecticides; ratios were higher in Bt maize relative to the sprayed non-Bt control.

Conclusions/Significance

Overall, we find no uniform effects of Bt cotton, maize and potato on the functional guilds of non-target arthropods. Use of and type of insecticides influenced the magnitude and direction of effects; insecticde effects were much larger than those of Bt crops. These meta-analyses underscore the importance of using controls not only to isolate the effects of a Bt crop per se but also to reflect the replacement of existing agricultural practices. Results will provide researchers with information to design more robust experiments and will inform the decisions of diverse stakeholders regarding the safety of transgenic insecticidal crops.     

Cheaper is not always worse: strongly protective isolates of a defensive symbiont are less costly to the aphid host

Defences against parasites are typically associated with costs to the host that contribute to the maintenance of variation in resistance. This also applies to the defence provided by the facultative bacterial endosymbiont Hamiltonella defensa, which protects its aphid hosts against parasitoid wasps while imposing life-history costs. To investigate the cost–benefit relationship within protected hosts, we introduced multiple isolates of H. defensa to the same genetic backgrounds of black bean aphids, Aphis fabae, and we quantified the protection against their parasitoid Lysiphlebus fabarum as well as the costs to the host (reduced lifespan and reproduction) in the absence of parasitoids. Surprisingly, we observed the opposite of a trade-off. Strongly protective isolates of H. defensa reduced lifespan and lifetime reproduction of unparasitized aphids to a lesser extent than weakly protective isolates. This finding has important implications for the evolution of defensive symbiosis and highlights the need for a better understanding of how strain variation in protective symbionts is maintained.     

Handling time, prey preference, and functional response for Chrysoperla rufilabris in the laboratory

The predaceous larvae of Chrysoperla rufilabris (Burmeister) exhibited some success-motivated searching, particularly when feeding on Heliothis virescens (F.) eggs, but handling time did not decrease with experience. Handling time for H. virescens larvae was more than twice that for eggs. H. virescens larvae were preferred to cotton aphids (Aphis gossypii (F.)) while aphids were preferred to H. virescens eggs. C. rufilabris larvae exhibited a linear functional response to the three prey types tested, over the prey densities tested.USDA, ARS, Retire. Present address: 200 Highland, College Station, TX 77840, USA     

Contemporary evolution of a Lepidopteran species,Heliothis virescens,in response to modern agricultural practices

Adaptation to human‐induced environmental change has the potential to profoundly influence the genomic architecture of affected species. This is particularly true in agricultural ecosystems, where anthropogenic selection pressure is strong. Heliothis virescens primarily feeds on cotton in its larval stages, and US populations have been declining since the widespread planting of transgenic cotton, which endogenously expresses proteins derived from Bacillus thuringiensis (Bt). No physiological adaptation to Bt toxin has been found in the field, so adaptation in this altered environment could involve (i) shifts in host plant selection mechanisms to avoid cotton, (ii) changes in detoxification mechanisms required for cotton‐feeding vs. feeding on other hosts or (iii) loss of resistance to previously used management practices including insecticides. Here, we begin to address whether such changes occurred in H. virescens populations between 1997 and 2012, as Bt‐cotton cultivation spread through the agricultural landscape. For our study, we produced an H. virescens genome assembly and used this in concert with a ddRAD‐seq‐enabled genome scan to identify loci with significant allele frequency changes over the 15‐year period. Genetic changes at a previously described H. virescens insecticide target of selection were detectable in our genome scan and increased our confidence in this methodology. Additional loci were also detected as being under selection, and we quantified the selection strength required to elicit observed allele frequency changes at each locus. Potential contributions of genes near loci under selection to adaptive phenotypes in the H. virescens cotton system are discussed.     

Impact of different levels of non-Bt cotton refuges on pest populations,bollworm damage,and Bt cotton production

The impact of structured strip row refugia (varying from 10% to 50%) in the Bt cotton crops JKCH1947Bt (producing one toxin, Cry1Ac) and MRC7017BGII (producing two toxins, Cry1Ac and Cry2Ab) on the pest complex and cotton yield was studied. During the cropping season (June 2008 to November 2008), sucking pest incidence was negligible. However, the incidences of spotted bollworm, Earias vittella, and the leafroller, Sylepta derogata, were high on the non-Bt cotton. The total cotton seed yield of the Bt crop plus the refuge decreased proportionately with respect to the increase in proportion of non-Bt cotton. Total cotton production decreased significantly when 40% non-Bt cotton was planted as refuge. These studies showed that a refuge of up to 30% non-Bt cotton in JKCH1947Bt and up to 20% non-Bt cotton in MRC7017Bt did not affect total seed cotton yield compared to 100% Bt cotton.     

Impact of Cotton Production Systems on Management of Hoplolaimus columbus

The effectiveness of selected cultural practices in managing the Columbia lance nematode, Hoplolaimus columbus, on cotton was evaluated in experiments in growers'' infested fields. The effects of planting date, cotton cultivar, treatment with the growth regulator mepiquat chloride, and destruction of cotton-root systems after harvest on cotton-lint yield and population densities of H. columbus were studied. The yield of cotton cultivar Deltapine 50 was negatively related (P = 0.054) to initial population density of H. columbus whereas the yield of Deltapine 90 was not affected by preplant density of this nematode, indicating tolerance in Deltapine 90. Reproduction of this nematode did not differ on the two cultivars. Planting date and treatment with the growth regulator mepiquat chloride did not influence cotton yield in a consistent manner. Application of mepiquat chloride suppressed (P ≤ 0.05) numbers of Columbia lance nematode, although there was an interaction (P ≤ 0.05) with cultivar and year. Early vs. late destruction of cotton-root systems did not impact population densities of this nematode either year, and had no impact on the subsequent cotton crop. The nematicide fenamiphos increased (P ≤ 0.03) cotton yield when H. columbus numbers exceeded the damage threshold.     

Transmission of Pandora neoaphidis in the presence of co-occurring arthropods

Transmission of the entomopathogenic fungus Pandora neoaphidis to the nettle aphid Microlophium carnosum was assessed in the presence of arthropods that co-exist with the fungus within the habitat but do not compete for aphid hosts. The presence of a parasitoid significantly enhanced transmission, and transmission rates were similar for both enemy and non-enemy parasitoids. Although herbivory of nettle leaves by Peacock butterfly (Inchis io) caterpillars indirectly reduced the number of M. carnosum by >30% due to a reduction in leaf area for feeding, the addition of I. io significantly increased transmission of P. neoaphidis in the remaining aphids. It is likely that enhanced transmission in the presence of A. rhopalosiphii and I. io is due to disturbance and subsequent movement of the aphid, resulting in contact with conidia deposited on the leaf surface. The presence and impact of co-occurring arthropods should be taken into consideration when assessing the transmission of fungal entomopathogens.     

Cereal Cyst Nematode (Heterodera avenae) on Oats. II. Early Root Development and Nematode Tolerance

The effect of Heterodera avenae infestation on early seminal and lateral root growth was examined in four oat genotypes differing in tolerance to H. avenae. Recently emerged seminal roots were inoculated with a range of H. avenae larval densities, then transferred a hydroponic system to remove the effect of later nematode penetration on root development. Intolerance to H. avenae was assessed in terms of impairment of seminal root extension resulting in fewer primary lateral roots emerging from the seminal root below the zone of juvenile penetration. Tolerant plants infested with H. avenae had longer lateral root systems than infested intolerant plants. The decline in lateral root growth below the penetration zone was partly offset by increased growth above. This did not contribute to tolerance, however, as there were no differences between cultivars for this feature. Nematodes induced earlier nodal root emergence in all cultivars. Nodal root development was most advanced on the most tolerant cultivar.