细菌冰核基因的应用研究

细菌冰核基因的应用研究已成为生物冰核领域的研究热点,研究涉及细菌细胞表面展示、促冻杀虫、报告基因、病原微生物高敏检测、作物抗寒育种等多个领域,显示良好应用前景。通过对国外在该方面的研究现状的综述,和我们在冰核基因促冻杀虫研究方面重要进展的介绍,对今后我们拟开展的这一研究工作进行了展望。     

The Use of High-resolution Infrared Thermography (HRIT) for the Study of Ice Nucleation and Ice Propagation in Plants

Freezing events that occur when plants are actively growing can be a lethal event, particularly if the plant has no freezing tolerance. Such frost events often have devastating effects on agricultural production and can also play an important role in shaping community structure in natural populations of plants, especially in alpine, sub-arctic, and arctic ecosystems. Therefore, a better understanding of the freezing process in plants can play an important role in the development of methods of frost protection and understanding mechanisms of freeze avoidance. Here, we describe a protocol to visualize the freezing process in plants using high-resolution infrared thermography (HRIT). The use of this technology allows one to determine the primary sites of ice formation in plants, how ice propagates, and the presence of ice barriers. Furthermore, it allows one to examine the role of extrinsic and intrinsic nucleators in determining the temperature at which plants freeze and evaluate the ability of various compounds to either affect the freezing process or increase freezing tolerance. The use of HRIT allows one to visualize the many adaptations that have evolved in plants, which directly or indirectly impact the freezing process and ultimately enables plants to survive frost events.     

Photoperiodic and Thermal Regulation of Development and Cold Hardiness in Larvae of the Clover Leaf Weevil, Hypera punctata

Effects of photoperiod and temperature on the development and cold hardiness were investigated in larvae of Hypera punctata. At a relatively low temperature (15°C), the larvae fed less and developed more slowly under a 12L:12D (SD) photoperiod than under a 16L:8D photoperiod (LD). SD larvae had lower gut weight against the whole body weight and lower supercooling point (SCP) than the LD counterparts for the same instar and same body weight. This was because the larval SCP is markedly affected by the quantity of the gut content. Laboratory experiments indicated that the low temperature mortality of this larvae occurred mainly due to freezing irrespective of the photoperiod and temperature, suggesting that the lower lethal temperature (LLT) depends on the supercooling ability of larvae. The SD larvae tended to have a lower SCP and hence a lower LLT than the LD counterparts at 15 or 10°C, unlike at 20°C. Thus, the slower larval development under SD conditions at relatively low temperatures may prevent larvae from reaching the later instar, which have a higher SCP and thus less cold tolerance, during the coldest season. The suppressed feeding activity under SD conditions would lower the SCP, thereby reducing the possibility of lethal tissue freezing. Such a photoperiodic and thermal regulation of the larval development and the supercooling ability appear to represent adaptive mechanisms for winter survival in this beetle.     

上海地区红棕象甲的耐寒性研究

红棕象甲Rhynchophorus ferrugineus (Olivier)是我国危害棕榈科植物的重要入侵害虫, 为探明其越冬抗寒性, 利用过冷却点测定仪测定了红棕象甲上海种群不同虫态的过冷却点, 在低温箱内测定了该虫不同虫态的耐寒性, 然后结合田间越冬模拟试验、气象资料和寄主分布情况, 初步确定了红棕象甲在我国的越冬北界。结果表明： 红棕象甲过冷却点随虫态的发育程度的升高而下降, 卵、1龄幼虫、5龄幼虫、9龄幼虫和成虫的平均过冷却点分别为-5.92, -6.42, -7.19, -7.43和-11.84℃, 过冷却点由高到低的顺序依次为: 卵>幼虫>成虫。在6, 24, 48和72 h 4个时间处理下, 各虫态在低温与存活率之间呈显著或极显著的logistic回归, 半致死温度（Ltemp₅₀）均随处理时间的延长而上升, 不同虫态在处理72 h 后, 卵、1龄幼虫、5龄幼虫、9龄幼虫和成虫之间的Ltemp₅₀分别为1.61, -1.67, -2.39, -2.40和-0.40℃, 各虫态耐寒性由弱到强的顺序依次为: 卵<成虫<幼虫。红棕象甲不同发育阶段的过冷却点与其耐寒性并不完全相关, 幼虫和成虫均可能是该虫的越冬虫态。连续两年的田间模拟越冬试验表明, 在上海地区, 红棕象甲的幼虫和成虫的越冬存活率均在60％以上, 说明红棕象甲在上海地区是可以越冬的。根据这些结果, 结合寄主分布情况, 初步将红棕象甲在我国的越冬北界定于北纬35°附近, 即1月份0℃等温线左右。     

河南新乡小麦区荻草谷网蚜的冬季耐寒性研究

为给荻草谷网蚜Sitobion miscanthi的准确预测预报提供理论依据,本研究利用过冷却点(Supercooling point,SCP)、低温暴露死亡率和冷识别温度(80％死亡率时的温度)等昆虫耐寒性指标评价低温条件下不同发育阶段该虫的耐寒能力.结果表明,荻草谷网蚜各发育阶段的SCP变化范围为-27.10～-12.23℃,SCP为1龄若蚜>2龄若蚜>有翅孤雌成蚜>无翅孤雌成蚜>4龄若蚜>3龄若蚜,其中1龄和2龄若蚜的SCP均显著高于3龄和4龄的SCP(P<0.01),3龄和4龄若蚜的SCP无显著性差异(P>0.05),无翅孤雌成蚜与其它发育阶段蚜虫的SCP均无显著性差异(P>0.05);两种虫态的低温存活率和冷识别温度分析表明,在-10～-8℃不同低温下,荻草谷网蚜1龄若蚜低温存活率均显著高于初羽化有翅成蚜的低温存活率(P<0.05),1龄若蚜和初羽化有翅成蚜的冷识别温度分别为-8.5℃和-7.9℃;两种虫态在0℃经过4 h冷驯化后,1龄若蚜和初羽化有翅成蚜在2 h的存活率提升最高,分别为62％和64％,均显著高于无冷驯化的对照组(P<0.05).本研究结果充分说明了荻草谷网蚜具有极强的耐低温适应能力,结合河南新乡近5年冬季极端低温数据,本文推测该虫能在冬季极端低温高于其冷识别温度的年份在当地越冬.     

The Ability of Coptera haywardi (Ogloblin) (Hymenoptera: Diapriidae) to Locate and Attack the Pupae of the Mediterranean Fruit Fly, Ceratitis capitata (Wiedemann) (Diptera: Tephritidae), under Seminatural Conditions

In Latin America, the diapriid Coptera haywardi (Ogloblin) attacks the pupae of tephritid fruit flies. Anastrepha spp. are among its natural hosts, but in the laboratory it also develops in the exotic Mediterranean fruit fly, Ceratitis capitata (Wiedemann). Field cage tests demonstrated that C. haywardi could locate and parasitize Mediterranean fruit fly pupae under seminatural conditions as found in a Guatemalan coffee plantation. A mean of 18.3% of the pupae buried artificially at depths of 5 mm were parasitized by C. haywardi, while those buried at 15 mm suffered 3.2% parasitism. In a laboratory experiment, larvae that buried themselves to pupate were not significantly more likely to be parasitized than artificially buried pupae, although they may have left a physical or chemical trail that betrayed their presence. Thus, the artificial burial of pupae is unlikely to grossly underestimate C. haywardi efficacy in the field. Another field cage test found that mortality levels due to unsuccessful parasitoid attacks were similar to those resulting from successful parasitism. Thus, the actual effect of a mass-release might be considerably greater than that suggested from parasitism data alone. The results are considered sufficiently positive to encourage further testing of C. haywardi as a biological control agent of the Mediterranean fruit fly.     

Biological control of an exotic scale, Orthezia insignis Browne (Homoptera: Ortheziidae), saves the endemic gumwood tree, Commidendrum robustum (Roxb.) DC. (Asteraceae) on the island of St. Helena

St. Helena is a small South Atlantic island with a highly degraded, but internationally significant, terrestrial flora. The 2500 remaining gumwoods, Commidendrum robustum, are an important part of this remnant flora. In 1991, a South American scale insect, Orthezia insignis, was identified attacking the gumwood trees. By 1993, severe infestations had killed over 100 trees. If the number of dead trees had continued to increase exponentially, all 2500 trees would have died by 1995. This outcome was likely given abundant alternative host plants, and lack of natural enemies of O. insignis. Host range tests showed that the other members of the endemic genus Commidendrum were also at risk. The coccinellid predator, Hyperaspis pantherina, was released on St. Helena in 1993. H. pantherina numbers increased from 1994, coinciding with a 30× decrease in scale numbers. Scale outbreaks have not been reported on St Helena since 1995, and culturing of H. pantherina was discontinued because insufficient O. insignis prey could be found in the field. Extensive blackening from sooty molds on the surviving trees in 1995 suggested that the predator was effective just in time to prevent death of most trees. H. pantherina appears to have saved the field population of a rare endemic plant from extinction.     

Field Releases of the Predatory Mite Neoseiulus fallacis (Acari: Phytoseiidae) in Canada, Monitored by Pyrethroid Resistance and Allozyme Markers

The predacious phytoseiid mite Neoseiulus fallacis (Garman) is an important agent for the biological control of spider mites in deciduous fruit orchards in North America and Canada. It would be helpful to monitor the fate of released individuals to improve the results of introductions of the predators in biological control trials. We have used two types of genetic markers, pyrethroid resistance and allozymes, for indirect estimation of the survival of N. fallacis introduced in an apple orchard in Ontario, Canada. Mite samples were submitted to toxicological tests. The polymorphism of four enzymes was studied in individual females using an isoelectric focusing technique. A mite sample was taken from the field, mass-reared in the laboratory, and selected for permethrin resistance. This strain was released on several apple trees treated with permethrin, and mite samples were collected from the same trees 10 to 90 days later. The genetic composition and the insecticide resistance level of this sample were compared to those of two other samples from trees where mites had not previously been released, either in the same orchard or in a neighboring block. A control susceptible strain was compared using mites collected earlier from trees on the same site but outside the present experiment. The mites collected from the release trees and those from the strain used for the releases were found to be genetically closely related, as judged from a small genetic distance, and from similar levels of insecticide resistance in both samples. The control samples from the nonrelease trees were genetically distant from these and displayed low resistance levels. These results indicate that the released genotypes established and persisted in the release trees for the period of the experiment. The utility of the two approaches in assessing the fate of released natural enemies is discussed.     

The effect of larval predators Thanasimus dubius (Coleoptera: Cleridae), produced by an improved system of rearing, against the southern pine beetle Dendroctonus frontalis (Coleoptera: Curculionidae)

The southern pine beetle, Dendroctonus frontalis (Coleoptera: Curculionidae) (SPB), is known to be a major bark beetle pest of pines throughout the southeastern United States. A common predator of bark beetles, Thanasimus dubius (Coleoptera: Cleridae), has been suggested to play a prevalent role on SPB dynamics. Evaluations of T. dubius have been limited by rearing methods; an artificial diet for larval T. dubius exists, and preservatives such as sorbic acid could help to maximize diet shelf-life and enhance the efficiency of the rearing system. The effects of sorbic acid at different concentrations (0%, 0.1% and 0.2%) in the larval diet for T. dubius were measured, and the effects of increased feeding time intervals (2-3 vs. 5 days) on predator performance evaluated. In addition, an experimental bioassay was conducted where newly hatched T. dubius larvae were released at four densities (0, 50, 100, and 200 per log) on pine logs infested by SPB. Sorbic acid in the diet reduced female fecundity (by 20-40%), but did not affect adult T. dubius size or longevity. However, using this preservative may not be necessary because it had no effect on the overall efficiency of the rearing system, while refreshing the larval diet every 5 days (compared with 2-3 days) did improve its efficiency, even without sorbic acid. The release of larval T. dubius resulted in a highly significant effect on the SPB ratio of increase (RI). This experiment was facilitated by the improvements in our rearing methods.     

Co-induction of DNA relaxation and synthesis of DnaK and GroEL proteins inEscherichia coli by expression of LetD (CcdB) protein, an inhibitor of DNA gyrase encoded by the F factor

We examined the influence of overexpression of LetD (CcdB) protein, an inhibitor of DNA gyrase encoded by the F factor ofEscherichia coli, on DNA supercoiling and induction of heat shock proteins. Cells were transformed with a plasmid carrying the structural gene for LetD protein under control of thetac promoter, and LetD protein was induced by adding isopropyl-d-thiogalactopyranoside (IPTG) to the culture medium. Analysis by agarose gel electrophoresis in the presence of chloroquine revealed relaxation of plasmid DNA in cells depending on the concentration of IPTG employed for induction. Protein pulse-labeling experiments with [³⁵S]methionine and cysteine revealed that synthesis of DnaK and GroEL proteins was also induced by IPTG, and concentrations necessary for DNA relaxation and induction of the heat shock proteins were much the same. Expression of mutant LetD protein lacking two amino acid residues at the C-terminus induced neither DNA relaxation nor the synthesis of DnaK and GroEL proteins. Induction of wild-type LetD protein but not mutant LetD protein markedly enhanced synthesis of ³². We interpret these results to mean that DNA relaxation in cells caused by the expression of LetD protein induces heat shock proteins via increased synthesis of ³².