Transgenic sugarcane plants expressing high levels of modified cry1Ac provide effective control against stem borers in field trials

To improve transgene expression level, we synthesized a truncated insecticidal gene m-cry1Ac by increasing its GC content from 37.4 to 54.8%, based on the codon usage pattern of sugarcane genes, and transferred it into two sugarcane cultivars (ROC16 and YT79-177) by microprojectile bombardment. The integration sites and expression pattern of the transgene were determined, respectively, by Southern, northern and western blot analyses. The transgenic sugarcane lines produced up to 50?ng Cry1Ac protein per mg soluble proteins, which was about fivefold higher than that produced by the partially modified s-cry1Ac (GC%?=?47.5%). In greenhouse plant assay, about 62% of the transgenic lines exhibited excellent resistance to heavy infestation by stem borers. In field trials, the m-cry1Ac transgenic sugarcane lines expressing high levels of Cry1Ac were immune from insect attack. In contrast, expression of s-cry1Ac in transgenic sugarcane plants resulted in moderately decreased damages in internodes (0.4-1.7%) and stalks (13.3-26.7%) in comparison with the untransformed sugarcane controls, which showed about 4 and 26-40% damaged internodes and stalks, respectively. Significantly, these transgenic sugarcane lines with high levels of insect resistance showed similar agronomic and industrial traits as untransformed control plants. Taken together, the findings from this study indicate a promising potential of engineering an insect-resistant gene to tailor its protein expression levels in transgenic sugarcane to combat insect infestations.     

F2 screen for resistance to a Bacillus thuringiensis-maize hybrid in the sugarcane borer (Lepidoptera: Crambidae)

A novel F2 screening technique was developed for detecting resistance in sugarcane borer, Diatraea saccharalis (F.), to transgenic Bacillus thuringiensis (Bt)-maize expressing the Cry1Ab insecticidal protein. The F2 screening method involved (i) collecting larvae from maize fields; (ii) establishing two-parent families; (iii) screening F2 neonates for survival on Bt-maize leaf tissues; and (iv) confirming resistance on commercial Bt-maize plants. With the F2 screening method, 213 iso-line families of D. saccharalis were established from field collections in northeast Louisiana, USA and were screened for Bt resistance. One family was confirmed to carry a major Bt resistance allele(s). In a laboratory bioassay, larval mortality of the Bt-resistant D. saccharalis on Bt-maize leaf tissues was significantly lower than that of a Bt-susceptible strain. This Bt-resistant D. saccharalis population is the first corn stalk borer species that has completed larval development on commercial Bt-maize. The F2 screening protocol developed in this study could be modified for detecting Bt resistance alleles in other similar corn stalk borers, such as the European corn borer, Ostrinia nubilalis (Hübner), and the southwestern corn borer, D. grandiosella Dyar.     

Sugarcane borer (Lepidoptera: Crambidae) management threshold assessment on four sugarcane cultivars

This research assesses the potential for using different economic injury thresholds in management of a key insect pest on susceptible and resistant commercially produced cultivars of sugarcane (Saccharum spp. hybrids). In a 2-yr sugarcane borer, Diatraea saccharalis (F.) (Lepidoptera: Crambidae), study involving four commercially produced sugarcane cultivars and four insecticide treatment thresholds, 'LCP 85-384' and 'HoCP 91-555' were the most susceptible based on percentage of bored internodes compared with the more resistant 'HoCP 85-845' and 'CP 70-321'. In 2001, the 10% infested stalks threshold was not as effective as the 5% early season-10% late season and 5% full season for HoCP 91-555. Based on D. saccharalis injury under natural infestation conditions, susceptible cultivars seem to require a lower infestation threshold than the more resistant cultivars to achieve adequate injury reduction. Among yield components, only the theoretical recoverable sugar per stalk was significantly increased by applying insecticides. With the resistant HoCP 85-845, differences were not detected for percentage of bored internodes among treated versus untreated management regimes. The resistant HoCP 85-845 had higher levels of fiber in our study; however, no clear pattern on resistance mechanisms was established, because the resistant cultivar CP 70-321 had comparatively low levels of fiber. The development of cultivar-specific thresholds is expected to lower the amount of insecticide used for D. saccharalis management in the sugarcane industry, reduce selection pressure, and delay the development of insecticide resistance.     

Evaluation of lectin-expressing transgenic sugarcane against stalkborers (Lepidoptera: Pyralidae): effects on life history parameters

The impact of snowdrop lectin (Galanthus nivalis agglutinin, GNA) expressed in transgenic sugarcane on life history parameters of Mexican rice borer [Eoreuma loftini (Dyar)] and sugarcane borer [Diatraea saccharalis (F.)] (both Lepidoptera: Pyralidae) was evaluated. In the laboratory, lyophilized sugarcane leaf sheath tissue was incorporated in a meridic diet resulting in a GNA concentration of 0.47% of total protein, and used for insect bioassays over two successive generations. Deleterious effects of GNA were not observed on survival, weight, and developmental periods of larvae and pupae, nor on adult fecundity and egg viability of D. saccharalis. Moreover, in the first generation, addition of transgenic sugarcane tissue to the diet enhanced larval growth in D. saccharalis resulting in higher larval and pupal weight compared with diet with nontransgenic sugarcane, but this effect was not observed in the second generation. In contrast, larval survival, percent adult emergence, and female fecundity of E. loftini were significantly reduced when fed transgenic sugarcane diet compared with nontransgenic sugarcane diet. In addition, a substantial reduction of female pupal weight of E. loftini was observed in the second generation. For both species, the only consistent effect of GNA in both generations was a reduction in adult female longevity. Life table parameters showed that GNA at the level found in the transgenic diet negatively affected development and reproduction of E. loftini, whereas it had a nil to positive effect on development and reproduction of D. saccharalis.     

Laboratory and field evaluation of Beauveria bassiana against sugarcane stalkborers (Lepidoptera: Pyralidae) in the lower Rio Grande Valley of Texas

Of approximately 18,200 ha planted to sugarcane in south Texas, only approximately 80 ha (<0.5%) are treated with insecticides because this type of control is widely regarded as ineffective against stalkboring pyralids, the key pests of sugarcane. Therefore, nonchemical control measures, such as resistant varieties and biological controls, must be evaluated to mitigate the losses caused by stalkborers. We performed laboratory and field evaluations on the use of Beauveria bassiana (Balsamo) Vuillemin (Deuteromycotina: Hyphomycetes) (strain GHA) against the Mexican rice borer, Eoreuma loftini (Dyar) (Lepidoptera: Pyralidae), which causes damage in south Texas estimated at between 10 and 20 million dollars annually. We also performed bioassays against the sugarcane borer, Diatraea saccharalis (F.) (Pyralidae), the key pest in other sugarcane growing areas. In the bioassays, E. loftini was substantially more susceptible to B. bassiana than D. saccharalis, based on both 5-d LD50 values and survival times. A commercial oil-based formulation of B. bassiana was evaluated in the field using the following treatments: oil alone (control), B. bassiana + oil, and B. bassiana + Silwet L-77 carrier at an application rate of 5 x 10(13) spores per hectare. Neither numbers of E. loftini per stalk, nor stalk damage (approximately 20% bored internodes) were significantly affected by treatment. The application of B. bassiana + Silwet significantly affected the numbers of internodes showing high damage, but not those with low or medium damage. Analysis of yield data and juice quality showed no significant treatment effects. We conclude that the application of Beauveria + Silwet offers the best chances for reducing damage caused by E. loftini of those treatments tested. However, reductions in insect incidence or damage did not result in measurable increases in yield or sugar quality, probably because of insufficient coverage. Effective control of stalkboring pyralids in sugarcane using B. bassiana will likely require improvements in delivery technology.     

Water stress augments silicon-mediated resistance of susceptible sugarcane cultivars to the stalk borer Eldana saccharina (Lepidoptera: Pyralidae)

Silicon (Si) can improve resistance of plants to insect attack and may also enhance tolerance of water stress. This study tested if Si-mediated host plant resistance to insect attack was augmented by water stress. Four sugarcane cultivars, two resistant (N21, N33) and two susceptible (N26, N11) to Eldana saccharina Walker were grown in a pot trial in Si-deficient river sand, with (Si+) and without (Si-) calcium silicate. To induce water stress, irrigation to half the trial was reduced after 8.5 months. The trial was artificially infested with E. saccharina eggs after water reduction and harvested 66 days later. Silicon treated, stressed and non-stressed plants of the same cultivar did not differ appreciably in Si content. Decreases in numbers of borers recovered and stalk damage were not associated with comparable increases in rind hardness in Si+ cane, particularly in water-stressed susceptible cultivars. Overall, Si+ plants displayed increased resistance to E. saccharina attack compared with Si- plants. Borer recoveries were significantly lower in stressed Si+ cane compared with either stressed Si- or non-stressed Si- and Si+ cane. Generally, fewer borers were recovered from resistant cultivars than susceptible cultivars. Stalk damage was significantly lower in Si+ cane than in Si- cane, for N21, N11 and N26. Stalk damage was significantly less in Si+ combined susceptible cultivars than in Si- combined susceptible cultivars under non-stressed and especially stressed conditions. In general, the reduction in borer numbers and stalk damage in Si+ plants was greater for water-stressed cane than non-stressed cane, particularly for susceptible sugarcane cultivars. The hypothesis that Si affords greater protection against E. saccharina borer attack in water-stressed sugarcane than in non-stressed cane and that this benefit is greatly enhanced in susceptible cultivars is supported. A possible active role for soluble Si in defence against E. saccharina is proposed.     

Molecular biology of wound-inducible proteinase inhibitors in plants

Abstract. The techniques of molecular biology are being employed to investigate at the gene level the systemically mediated, wound-induced accumulation of two defensive proteinase inhibitor proteins in plant leaves. These techniques have added a new dimension to biochemical and physiological studies already underway to understand the mechanism of induction by wounding. The acquisition of cDNAs from the RNAs coding for the two inhibitors facilitated studies of mRNA synthesis in leaves in response to wounding, and provided probes to obtain wound-inducible proteinase inhibitor genes from tomato ( Lycopersicon esculentum ) and potato (Solarium tuberosum) genomes. Successful transformations of tobacco plants with fused genes, containing the 5' and 3' regions of the inhibitor genes with the open reading frame of the chloramphenicol acelyltransferase ( cat ) gene, have provided a wound-inducible chloramphenicol acetyltransferase (CATase) activity with which to seek cis- and transacting elements that regulate wound-inducibility to help to understand the interaction of cytoplasmic and nuclear components of the intracellular communication systems that activate the proteinase inhibitor genes in response to wounding by insect pests.     

Feasibility of using an alternative larval host and host plants to establish Cotesia flavipes (Hymenoptera: Braconidae) in the temperate Louisiana sugarcane ecosystem

Attempts to establish Cotesia flavipes Cameron (Hymenoptera: Braconidae) in Louisiana sugarcane fields to control the sugarcane borer, Diatraea saccharalis (F.) (Lepidoptera: Crambidae) have been unsuccessful. Experiments were conducted to investigate the feasibility of using an alternative larval host and host plants to overcome barriers preventing establishment. In addition, we evaluated C. flavipes' ability to search for D. saccharalis in sugarcane without above-ground internodes. Diatraea evanescens Dyar (Lepidoptera: Crambidae) was investigated as an alternative host for C. flavipes. Cotesia flavipes was reared for five generations on D. evanescens without any indication of diminishing fitness as measured by days to parasite pupation and average cocoon mass weight. However, there was a significant reduction in percent parasitism, cocoon mass weight, and percent emergence when C. flavipes parasitized D. evanescens as compared with D. saccharalis, resulting in a 75% reduction in the gross reproductive rate (R(0)). Greenhouse studies indicated little difference in parasitism of D. saccharalis on the weed hosts johnsongrass, Sorghum halepense (L.), and vaseygrass, Paspalum urvillei Steud. However, when planted as refuge plots, we found it difficult to establish infestations of D. saccharalis in either of these hosts, or in two energy sugarcanes. After 3 yr of infesting host plants and releasing parasitoids only one parasitized D. saccharalis larvae was recovered within the johnsongrass refuge. Diatraea evanescens readily established in vaseygrass; however, these larvae appear inaccessible to C. flavipes. In contrast, parasitism of D. saccharalis by C. flavipes infesting young sugarcane was 30%.     

Ant predation of larval Diatraea saccharalis Fab. (Lep., Crambidae) in new sugarcane in Brazil

Abstract: Ant predation of natural field populations of larval sugarcane borers, Diatraea saccharalis , was evaluated by using insecticidal check techniques, and through survivorship studies of artificial high-density infestations, in plantations in Brazil. Areas of ant suppression had higher levels of D. saccharalis than unsuppressed areas, although both types of areas normally had extremely low D. saccharalis populations. Artifical infestations of D. saccharalis yielded significantly lower larval survivorship in unsuppressed areas than in suppressed areas. Fire ants ( Solenopsis spp.) were always the most abundant ant. This study has demonstrated that ants are important pest predators in Brazilian sugarcane fields.     

人细胞周期蛋白D1/CDK4基因的真核表达及生物活性鉴定

通过生物工程获得人重组细胞周期蛋白 (cyclinD1 )及细胞周期蛋白激酶CDK4蛋白 ,作为抗癌药物筛选的分子靶点 .从人HL 6 0细胞中获得细胞周期蛋白D1 CDK4基因的cDNA ,先克隆至pGEMT Easy载体上 ,再经重组构建供体质粒pFastBac D1和pFastBac CDK4 .重组供体质粒转化感受态DH1 0Bac细胞 ,挑取确证为白色克隆的菌落振荡培养 ,分离制备高纯度杆粒DNA .以重组病毒适量感染昆虫细胞Tn 5B1 4 ,利用Bac to Bac杆状病毒表达系统在昆虫细胞Tn 5B1 4 (Hi5 )中表达相应的重组蛋白 .应用昆虫杆状病毒表达系统 (Bac to Bac)在昆虫细胞Tn 5B1 4中分别高效表达了人细胞周期蛋白D1和CDK4蛋白 .SDS PAGE分析表明 ,表达量占细胞可溶性蛋白质的 2 0 %左右 ,表达产物经Ni2 + NTA亲和层析纯化后纯度达 85 %以上 .研究表明 ,昆虫细胞表达的细胞周期蛋白D1和CDK4蛋白能促进Rb蛋白的磷酸化 ,具有生物活性 .成功构建了细胞周期蛋白D1及CDK4真核杆状病毒表达载体 ,并且在昆虫细胞中正确表达了具有生物活性的细胞周期蛋白D1及CDK4融合蛋白 .     

Abscisic acid and jasmonic acid activate wound-inducible genes in potato through separate, organ-specific signal transduction pathways

Mechanical damage to leaf tissue causes an increase in abscisic acid (ABA) which in turn activates the biosynthesis of jasmonic acid (JA). The resulting higher endogenous JA levels subsequently activate the expression of wound-inducible genes. This study shows that JA induces the expression of different sets of genes in roots and leaves of potato plants. When roots of intact plants were treated with JA, high levels of proteinase inhibitor II (pin2), cathepsin D inhibitor, leucine aminopeptidase and threonine deaminase mRNAs accumulated in the systemic leaves. However, in the treated roots, very low, if any, expression of these genes could be detected. In contrast, a novel, root-specific pin2 homologue accumulated in the JA-treated root tissue which could not be detected in leaves, either systemic or those directly treated with JA. Application of okadaic acid and staurosporine revealed that a protein phosphorylation step is involved in the regulation of this differential response. In leaves, a protein phosphatase is required for the JA-induced expression of pin2 and the other genes analysed. This phosphatase activity is not necessary for the JA-induced expression of a pin2 homologue in roots, suggesting the existence of different transduction pathways for the JA signal in these organs. The requirement of a protein phosphatase activity for JA-mediated gene induction has enabled identification of a JA-independent pathway for ABA induction of pin2 and the other wound-inducible genes. This alternative pathway involves a protein kinase, and appears to be selective for wound-inducible genes. Our data suggest the presence of a complex, organ-specific transduction network for regulating the effects of the plant hormones ABA and JA on gene expression upon wounding.     

Effects of entomopathogenic bacterium Photorhabdus temperata infection on the digestive enzymes of Diatraea saccharalis (Lepidoptera: Crambidae) larvae

This study investigated the effects of Photorhabdus temperata infection on the activities of digestive enzymes of the sugarcane stalk borer Diatraea saccharalis. Non-infected D. saccharalis larvae present a major alpha-amylase, several proteinases, three sucrose hydrolases and two alpha-glucosidases in their midgut. Analysis of these hydrolases by electrophoresis and "in gel" assays showed that the activities of all enzymes decreased following infection, with an initial decline observed 12 h after infection. The activities of alpha-glucosidases decreased by 50% twelve hours after infection, whereas, at this time, the alpha-galactosidase activities decreased by 70%. Interestingly, the animals died 48 h after infection, but approximately 5% of all the enzymes tested remained active in the midgut following host death. At this time, most of the cultivable native intestinal bacteria had died.     

Spatial pattern of parasitism in Diatraea saccharalis Fab. (Lep., Crambidae) populations at two different spatial scales in sugarcane fields in Brazil

Abstract:  The biological control of Diatraea saccharalis is regarded as one of the best examples of successful classical biological control in Brazil. Since the introduction of the exotic parasitoid, Cotesia flavipes , the decrease in D. saccharalis infestation in sugarcane fields has been attributed to the effectiveness of this agent. Native Tachinidae fly parasitoids ( Lydella minense and Paratheresia claripalpis ) have also been implicated in the success. Quantitative data confirming the actual contribution of these agents to the control of D. saccharalis are, however, rather scant. The purpose of this study was to investigate the spatial pattern of parasitism of these parasitoids in D. saccharalis populations at two large spatial scales (fields and zones). To investigate this subject, a large data set comprising information collected from a sugarcane mill located in the state of São Paulo, Brazil (São João sugarcane mill) was analysed. When regressions between the proportion parasitism against host density were computed, the percentage of significant regressions with either a positive or a negative slope was very small at both spatial scales for both parasitoid species. Regressing the densities of tachinid-parasitized hosts against host densities per field showed that these parasitoids presented a 'moderate aggregative' response to host densities, as 53.33% of the regressions were positively significant. Cotesia flavipes was 'weakly aggregated' on host densities at the field level, because only 33.33% of the regressions were positively significant. At the zone level, neither aggregative nor spatial proportion parasitism responses were evident for either parasitoid species due to the small percentage of significant regressions computed.     

Silicon impedes stalk penetration by the borer Eldana saccharina in sugarcane

Many plants grown in soils amended with silicon (Si) display increased levels of resistance to attack by insect herbivores. This study aimed to determine if Si treatment impeded Eldana saccharina Walker (Lepidoptera: Pyralidae) stalk penetration and subsequent damage, as well as borer mass gain, on the node and internode of a susceptible (N11) and a resistant (N33) sugarcane cultivar. Sugarcane [Saccharum spp. (Poaceae)] cultivars were grown in a pot trial in Si‐deficient river sand, with (Si+) and without (Si–) calcium silicate. Sugarcane was infested with 2‐week‐old E. saccharina larvae and harvested at four times, 24, 48, 72, and 96 h after infestation. Silicon‐treated plants showed significant increases in Si content compared to controls, and the external rind was significantly harder for Si+ cane than Si– cane. Silicon treatment significantly decreased borer penetration, stalk damage, and larval mass gain. The results are consistent with the hypothesis that Si contributes to sugarcane stalk borer resistance by impeding larval penetration. Silicon appears to contribute to the suppression of E. saccharina directly through reduced larval growth and feeding damage to the crop, and indirectly by delaying stalk penetration, resulting most likely in increased exposure time of young larvae to natural enemies, adverse climatic factors, or control measures that target young larvae (e.g., insecticides).     

Sampling for the sugarcane borer (Lepidoptera: Crambidae) on sugarcane in Louisiana.

A 3-yr study was conducted in 0.6- to 2.0-ha sugarcane fields throughout south Louisiana under varying sugarcane borer, Diatraea saccharalis (F.), density levels to determine the spatial dispersion of infestations and to develop a sequential sampling plan. Infestations of D. saccharalis were randomly dispersed. Infestation levels (percentage of stalks infested) ranged from 0.6 to 33.3%. Frequency distributions of the number of infested stalks indicated that the Poisson distribution best fit the data Tests of other distributions (negative binomial [aggregated], binomial [uniform], geometric, and hypergeometric) resulted in poorer fits. The sequential sampling plan devised, with lower and upper D. saccharalis infestation limits of 2 and 5% and 5 and 10%, required maximum average sample numbers of 7.1 and 5.5 (20-stalk samples), respectively, to make terminating management decisions. It is our assessment that implementation of these plans would decrease sampling effort by 50-60% when compared with sampling programs currently in use for D. saccharalis management decisions in Louisiana.