Genomic characterization of Ugandan smallholder farmer-preferred cassava varieties.

Understanding the genetic relationships among farmer-preferred cassava (Manihot esculenta Crantz) varieties is indispensable to genetic improvement efforts. In this study, we present a genetic analysis of 547 samples of cassava grown by 192 smallholder farmers, which were sampled at random within four districts in Uganda. We genotyped these samples at 287,952 single nucleotide polymorphisms using genotyping-by-sequencing and co-analyzed them with 349 cassava samples from the national breeding program in Uganda. The samples collected from smallholders consisted of 86 genetically unique varieties, as assessed using a genetic distance-based approach. Of these varieties, most were cultivated in only one district (30 in Kibaale, 19 in Masindi, 14 in Arua, and three in Apac), and only three were cultivated across all districts. The genetic differentiation we observed among farming districts in Uganda (mean fixation index [F ST] = .003) is similar to divergence observed within other countries. Despite the fact that none of the breeding lines were directly observed in farmer fields, genetic divergence between the populations was low (F ST = .020). Interestingly, we detected the presence of introgressions from the wild relative M. glaziovii Müll. Arg. on chromosomes 1 and 4, which implies ancestry with cassava breeding lines. Given the apparently similar pool of alleles in the breeding germplasm, it is likely that breeders have the raw genetic material they require to match the farmer-preferred trait combinations necessary for adoption. Our study highlights the importance of understanding the genetic makeup of cassava currently grown by smallholder farmers and relative to that of plant breeding germplasm.

Natural enemies delay insect resistance to Bt crops.

We investigated whether development of resistance to a Bt crop in the presence of a natural enemy would be slower than without the natural enemy and whether biological control, in conjunction with a Bt crop, could effectively suppress the pest population. Additionally, we investigated whether insecticide-sprayed refuges of non-Bt crops would delay or accelerate resistance to the Bt crop. We used a system of Bt broccoli expressing Cry1Ac, a population of the pest Plutella xylostella with a low frequency of individuals resistant to Cry1Ac and the insecticide spinosad, and a natural enemy, Coleomegilla maculata, to conduct experiments over multiple generations. The results demonstrated that after 6 generations P. xylostella populations were very low in the treatment containing C. maculata and unsprayed non-Bt refuge plants. Furthermore, resistance to Bt plants evolved significantly slower in this treatment. In contrast, Bt plants with no refuge were completely defoliated in treatments without C. maculata after 4-5 generations. In the treatment containing sprayed non-Bt refuge plants and C. maculata, the P. xylostella population was low, although the speed of resistance selection to Cry1Ac was significantly increased. These data demonstrate that natural enemies can delay resistance to Bt plants and have significant implications for integrated pest management (IPM) with Bt crops.

Bt crops producing Cry1Ac, Cry2Ab and Cry1F do not harm the green lacewing, Chrysoperla rufilabris.

The biological control function provided by natural enemies is regarded as a protection goal that should not be harmed by the application of any new pest management tool. Plants producing Cry proteins from the bacterium, Bacillus thuringiensis (Bt), have become a major tactic for controlling pest Lepidoptera on cotton and maize and risk assessment studies are needed to ensure they do not harm important natural enemies. However, using Cry protein susceptible hosts as prey often compromises such studies. To avoid this problem we utilized pest Lepidoptera, cabbage looper (Trichoplusia ni) and fall armyworm (Spodoptera frugiperda), that were resistant to Cry1Ac produced in Bt broccoli (T. ni), Cry1Ac/Cry2Ab produced in Bt cotton (T. ni), and Cry1F produced in Bt maize (S. frugiperda). Larvae of these species were fed Bt plants or non-Bt plants and then exposed to predaceous larvae of the green lacewing Chrysoperla rufilabris. Fitness parameters (larval survival, development time, fecundity and egg hatch) of C. rufilabris were assessed over two generations. There were no differences in any of the fitness parameters regardless if C. rufilabris consumed prey (T. ni or S. frugiperda) that had consumed Bt or non-Bt plants. Additional studies confirmed that the prey contained bioactive Cry proteins when they were consumed by the predator. These studies confirm that Cry1Ac, Cry2Ab and Cry1F do not pose a hazard to the important predator C. rufilabris. This study also demonstrates the power of using resistant hosts when assessing the risk of genetically modified plants on non-target organisms.

A critical assessment of the effects of Bt transgenic plants on parasitoids.

The ecological safety of transgenic insecticidal plants expressing crystal proteins (Cry toxins) from the bacterium Bacillus thuringiensis (Bt) continues to be debated. Much of the debate has focused on nontarget organisms, especially predators and parasitoids that help control populations of pest insects in many crops. Although many studies have been conducted on predators, few reports have examined parasitoids but some of them have reported negative impacts. None of the previous reports were able to clearly characterize the cause of the negative impact. In order to provide a critical assessment, we used a novel paradigm consisting of a strain of the insect pest, Plutella xylostella (herbivore), resistant to Cry1C and allowed it to feed on Bt plants and then become parasitized by Diadegma insulare, an important endoparasitoid of P. xylostella. Our results indicated that the parasitoid was exposed to a biologically active form of the Cy1C protein while in the host but was not harmed by such exposure. Parallel studies conducted with several commonly used insecticides indicated they significantly reduced parasitism rates on strains of P. xylostella resistant to these insecticides. These results provide the first clear evidence of the lack of hazard to a parasitoid by a Bt plant, compared to traditional insecticides, and describe a test to rigorously evaluate the risks Bt plants pose to predators and parasitoids.

Sequential transformation to pyramid two Bt genes in vegetable Indian mustard (Brassica juncea L.) and its potential for control of diamondback moth larvae.

Vegetable Indian mustard (Brassica juncea cv. "Green Wave") plants that control Plutella xylostella (diamondback moth) (DBM) were produced by introduction of one or two Bacillus thuringiensis (Bt) genes. A cry1Ac Bt gene associated with the nptII gene for kanamycin selection or a cry1C Bt gene with the hpt gene for hygromycin selection was introduced individually through Agrobacterium-mediated transformation of seedling explants. A cry1C line was then transformed with the cry1Ac gene to produce pyramided cry1Ac + cry1C plants. Sixteen cry1C, five cry1Ac, and six cry1Ac + cry1C plants were produced. PCR and Southern analyses confirmed the presence of the cry1C, cry1Ac or pyramided cry1Ac + cry1C genes in the Indian mustard genome. ELISA analysis showed that production of Bt proteins varied greatly among individual transgenic plants, ranging from undetectable to over 1,000 ng Bt/mg total soluble protein. The levels of the Bt proteins were correlated with the effectiveness of control of diamondback moth (DBM) larvae. Insect bioassays indicated that both the cry1C and cry1Ac plants were toxic to susceptible DBM. The cry1C plants also controlled Cry1A-resistant DBM while cry1Ac plants controlled Cry1C-resistant DBM, and the pyramided cry1Ac + cry1C plants effectively controlled all three types of DBM. These Bt-transgenic plants could be used either for direct control of DBM and other lepidopteran insect pests or for tests of "dead-end" trap crops as protection of high value non-transgenic crucifer vegetables such as cabbage.

Impact of single-gene and dual-gene Bt broccoli on the herbivore Pieris rapae (Lepidoptera: Pieridae) and its pupal endoparasitoid Pteromalus puparum (Hymenoptera: Pteromalidae).

Transgenic brassica crops producing insecticidal proteins from Bacillus thuringiensis (Bt) are being investigated as candidates for field release to control lepidopteran pests. Information on the potential impact of Bt brassica crops on pests and non-target natural enemies is needed as part of an environmental risk assessment prior to the commercial release. This first tier study provides insight into the tritrophic interactions among Bt broccoli plants, the herbivore Pieris rapae and its parasitoid Pteromalus puparum. We first evaluated the efficacy of three types of Bt broccoli plants, cry1Ac, cry1C and cry1Ac + cry1C, on different instars of P. rapae. Bt broccoli effectively controlled P. rapae larvae, although later instars were more tolerant. The efficacy of different Bt broccoli plants on P. rapae larvae was consistently cry1Ac > cry1Ac + cry1C > cry1C. When the parasitoid P. puparum developed in a P. rapae pupa (host) that had developed from Bt plant-fed older larvae, developmental time, total number and longevity of the P. puparum generated from the Bt plant-fed host were significantly affected compared with those generated from the non-Bt control plant-fed host. Simultaneously, negative effects on P. rapae pupae were found, i.e. pupal length, width and weight were significantly reduced after older P. rapae larvae fed on different Bt plants for 1 or 2 days. Cry1C toxin was detected using ELISA in P. rapae pupae after older larvae fed on cry1C broccoli. However, no Cry1C toxin was detected in newly emerged P. puparum adults developing in Bt-fed hosts. Only a trace amount of toxin was detected from entire P. puparum pupae dissected from the Bt plant-fed host. Moreover, no negative effect was found on the progeny of P. puparum developing from the Bt plant-fed host when subsequently supplied with a healthy host, P. rapae pupae. The reduced quality of the host appears to be the only reason for the observed deleterious effects on P. puparum. Our data suggest that the effects on P. puparum developing in Bt plant-fed P. rapae are mediated by host quality rather than by direct toxicity.

The cauliflower Or gene encodes a DnaJ cysteine-rich domain-containing protein that mediates high levels of beta-carotene accumulation.

Despite recent progress in our understanding of carotenogenesis in plants, the mechanisms that govern overall carotenoid accumulation remain largely unknown. The Orange (Or) gene mutation in cauliflower (Brassica oleracea var botrytis) confers the accumulation of high levels of beta-carotene in various tissues normally devoid of carotenoids. Using positional cloning, we isolated the gene representing Or and verified it by functional complementation in wild-type cauliflower. Or encodes a plastid-associated protein containing a DnaJ Cys-rich domain. The Or gene mutation is due to the insertion of a long terminal repeat retrotransposon in the Or allele. Or appears to be plant specific and is highly conserved among divergent plant species. Analyses of the gene, the gene product, and the cytological effects of the Or transgene suggest that the functional role of Or is associated with a cellular process that triggers the differentiation of proplastids or other noncolored plastids into chromoplasts for carotenoid accumulation. Moreover, we demonstrate that Or can be used as a novel genetic tool to induce carotenoid accumulation in a major staple food crop. We show here that controlling the formation of chromoplasts is an important mechanism by which carotenoid accumulation is regulated in plants.

beta-Carotene accumulation induced by the cauliflower Or gene is not due to an increased capacity of biosynthesis.

The cauliflower (Brassica oleracea L. var. botrytis) Or gene is a rare carotenoid gene mutation that confers a high level of beta-carotene accumulation in various tissues of the plant, turning them orange. To investigate the biochemical basis of Or-induced carotenogenesis, we examined the carotenoid biosynthesis by evaluating phytoene accumulation in the presence of norflurazon, an effective inhibitor of phytoene desaturase. Calli were generated from young seedlings of wild type and Or mutant plants. While the calli derived from wild type seedlings showed a pale green color, the calli derived from Or seedlings exhibited intense orange color, showing the Or mutant phenotype. Concomitantly, the Or calli accumulated significantly more carotenoids than the wild type controls. Upon treatment with norflurazon, both the wild type and Or calli synthesized significant amounts of phytoene. The phytoene accumulated at comparable levels and no major differences in carotenogenic gene expression were observed between the wild type and Or calli. These results suggest that Or-induced beta-carotene accumulation does not result from an increased capacity of carotenoid biosynthesis.

Bacillus thuringiensis protein production, signal transduction, and insect control in chemically inducible PR-1a/cry1Ab broccoli plants.

In an effort to develop a chemically inducible system for insect management, we studied production of Cry1Ab Bacillus thuringiensis (Bt) protein and control of the diamondback moth (DBM), Plutella xylostella L., in inducer-treated and untreated tissues of a broccoli line transformed with a PR-1a/cry1Ab expression cassette. Spraying leaves of these plants with the inducer acibenzolar-S-methyl (= 1,2,3 benzothiadiazole-7-thiocarboxylic acid-S-methyl-ester) (ASM) triggered expression of the cry1Ab gene and produced a high level of Cry1Ab protein within 2-3 days. Cry1Ab protein persisted in leaves for at least 8 weeks, providing prolonged protection from P. xylostella attack. Signals generated in inducer-treated leaves were transferred to untreated newly emerged leaves or heads, as seen by production of Cry1Ab protein and/or protection from insect damage in these plant parts. Signal transduction proceeded in an attenuated manner up to the sixth newly emerged leaf. No Cry1Ab protein was detectable by ELISA in uninduced young leaves, but small amounts of the protein were present in uninduced leaves older than 3 weeks and caused some insect mortality. Such basal expression of Bt genes without induction may favor the evolution of resistant insect populations and therefore limits the application of the PR-1a/cry1Ab system for insect management. However, the rapid production and steady maintenance of a high level of transgenic protein upon induction, the signal transduction observed, and the fact that the chemical inducer can be used in field conditions make the PR-1a promoter attractive for chemical regulation of other agriculturally or pharmaceutically important genes for which low expression in the absence of induction is not a concern.

Concurrent use of transgenic plants expressing a single and two Bacillus thuringiensis genes speeds insect adaptation to pyramided plants.

Transgenic plants expressing insecticidal proteins from the bacterium Bacillus thuringiensis (Bt) were grown on over 13 million ha in the United States and 22.4 million ha worldwide in 2004. Preventing or slowing the evolution of resistance by insects ("resistance management") is critical for the sustainable use of Bt crops. Plants containing two dissimilar Bt toxin genes in the same plant ("pyramided") have the potential to delay insect resistance. However, the advantage of pyramided Bt plants for resistance management may be compromised if they share similar toxins with single-gene plants that are deployed simultaneously. We tested this hypothesis using a unique model system composed of broccoli plants transformed to express different Cry toxins (Cry1Ac, Cry1C, or both) and a synthetic population of the diamondback moth (Plutella xylostella) carrying genes for resistance to Cry1Ac and Cry1C at frequencies of approximately 0.10 and 0.34, respectively. After 24-26 generations of selection in the greenhouse, the concurrent use of one- and two-gene plants resulted in control failure of both types of Bt plants. When only two-gene plants were used in the selection, no or few insects survived on one- or two-gene Bt plants, indicating that concurrent use of transgenic plants expressing a single and two Bt genes will select for resistance to two-gene plants more rapidly than the use of two-gene plants alone. The results of this experiment agree with the predictions of a Mendelian deterministic simulation model and have important implications for the regulation and deployment of pyramided Bt plants.

Evaluation of a chemically inducible promoter for developing a within-plant refuge for resistance management.

Chemically inducible production of Bacillus thuringiensis (Bt) toxins in transgenic plants may provide considerable benefits in preventing or delaying the evolution of insect resistance to Bt crops by creating within-plant temporal refuges. We examined the effect of inducible cry1Ab expression on survival of different genotypes (RR, RS, and SS) of diamondback moth, Plutella xylostella (L.), in transgenic broccoli, Brassica oleracea L., plants transformed with a PR-1a/cry1Ab expression cassette. Spraying leaves of these plants with the inducer acibenzolar-s-methyl [= benzo (1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester] (ASM) resulted in high levels of Bt toxin, and detached leaves from fully induced plants caused 100% mortality to all instars of P. xylostella SS and RS genotypes. When plants infested with larvae were treated with ASM, only a few larvae that were nearing completion of their development were able to survive the induction process. Signal transduction from ASM-treated leaves to new plant tissue also was evaluated using a larval assay. New foliage that emerged after plants were induced remained toxic to > or = 80% of RS larvae up to the fourth new leaf. In whole plant tests, however, induced plants remained protected from larval damage for > or = 3 wk. Uninduced PR-1a/cry1Ab plants seemed to produce low levels of Bt that were undetected by an enzyme-linked immunosorbent assay but that resulted in significant fitness costs for susceptible insects. The suitability of PR-1a/cry1Ab broccoli plants for insect resistance management and the requirements of an appropriate inducible promoter are discussed.

Transgenic plants expressing two Bacillus thuringiensis toxins delay insect resistance evolution.

Preventing insect pests from developing resistance to Bacillus thuringiensis (Bt) toxins produced by transgenic crops is a major challenge for agriculture. Theoretical models suggest that plants containing two dissimilar Bt toxin genes ('pyramided' plants) have the potential to delay resistance more effectively than single-toxin plants used sequentially or in mosaics. To test these predictions, we developed a unique model system consisting of Bt transgenic broccoli plants and the diamondback moth, Plutella xylostella. We conducted a greenhouse study using an artificial population of diamondback moths carrying genes for resistance to the Bt toxins Cry1Ac and Cry1C at frequencies of about 0.10 and 0.20, respectively. After 24 generations of selection, resistance to pyramided two-gene plants was significantly delayed as compared with resistance to single-gene plants deployed in mosaics, and to Cry1Ac toxin when it was the first used in a sequence. These results have important implications for the development and regulation of transgenic insecticidal plants.

Sensitivity of bacterial and fungal plant pathogens to the lytic peptides, MSI-99, magainin II, and cecropin B.

In vitro and leaf disk assays of bacterial and fungal plant pathogens were conducted using three cationic lytic peptides, MSI-99, magainin II (MII), and cecropin B (CB). Growth of bacterial organisms was retarded or completely inhibited by low concentrations of these lytic peptides. The peptides also significantly reduced germination of fungal spores and growth of mycelia; however, higher concentrations of peptides were needed to inhibit fungal growth compared with those needed to inhibit bacteria. The relative efficacy of the peptides depended on the microorganism tested, but CB was the most inhibitory to the majority of the bacteria and fungi assayed. MSI-99, a synthetic derivative of MII with increased positive charge, showed equal or two- to fivefold higher antibacterial activity compared to MII in the in vitro assays. MSI-99 was also superior to MII against the oomycete, Phytophthora infestans but was slightly inferior to MII in assays with the true fungi, Penicillium digitatum and Alternaria solani. In the leaf disk assays, pretreating spores of Alternaria solani and Phytophthora infestans with the peptides at concentrations as low as 10 microg per ml led to significant reductions in the size of early blight lesions and prevented development of any late blight lesions on tomato leaf disks. Our results from in vitro and leaf disk assays suggest that MSI-99 can be used as a transgene to generate tomato lines with enhanced resistance to bacterial and fungal diseases of this crop.