油菜素内酯与美极梅奇酵母复配对葡萄灰霉病的抑制作用

【背景】生防酵母具有繁殖速度快、抗逆性强和不产生抗生素等特点,但其对病害的防控效果易受环境影响,油菜素内酯(brassinosteroid,BR)能调控植物生长发育与逆境响应平衡,可有效抑制葡萄果实灰霉病的发生。【目的】本研究旨在明确BR与生防酵母复配对葡萄果实灰霉病的抑制效果及作用机制,为新型生防制剂的研制和应用提供理论依据。【方法】采用美极梅奇酵母P01C004 (Y)、酵母和BR (YBR)、酵母和BR抑制剂(YBZ)处理“红地球”葡萄果实,处理6 h后人工接种灰霉菌孢子悬液。灰霉菌接种7 d后评价BR与生防酵母复配对灰霉菌的防治效果,并检测其抗氧化酶活性和13种酚类物质含量,利用qRT-PCR检测不同处理对葡萄BR、白藜芦醇和抗病相关基因表达水平的影响。【结果】7 d时,与Y处理相比,YBR对葡萄果实灰霉病防治效率提高了23.64%,显著提高了“红地球”葡萄果实中PPO酶活。YBR显著提高了2 d时果实中绿原酸、原儿茶酸、咖啡酸、表儿茶素和芹菜素等酚类物质含量,激活了果实内多种酚类物质的快速合成。YBR在48 h时激活了果实BR信号转导途径,显著上调VvBZR1和VvPR1基因的表达水平,更好地维持植物免疫反应,提高果实对病原菌的防御力。【结论】BR与美极梅奇酵母复配能触发果实多种抗病防御机制,提高葡萄果实灰霉病的防治效率,在采后病害防控方面表现出良好的应用前景。     

Optimization for high-level expression of the Pichia guilliermondii recombinant inulinase in Pichia pastoris and characterization of the recombinant inulinase

The inulinase gene cloned from the marine-derived yeast Pichia guilliermondii strain 1 was expressed in Pichia pastoris X-33 and the conditions for overexpression of the inulinase were optimized. After the optimization of the conditions for production of the recombinant inulinase, 286.8 ± 5.4 U/ml and 8873 ± 55.3 U/mg of the recombinanat inulinase in the supernatant of the culture of 2-l fermentor were attained at 120 h of the fermentation and fermentation efficiency was 13.04 μg ± 0.4 of protein/ml/d. The recombinant inulinase was purified and characterized. The molecular weight of the purified recombinant inulinase was 57.6 kDa, which was higher than that of the native iunlinase. The optimal pH and temperature of the purified recombinant inulinase were 6.0 and 60 °C, respectively. Other biochemical characteristics of the purified recombinant inulinase were the same as those of the native inulinase produced by the marine-derived P. guilliermondii strain 1. The purified recombinant inulinase also had high exoinulinase activity. Therefore, the recombinant inulinase may have highly potential applications in food and pharmaceutical industies.     

Potential biocontrol activity of a strain of Pichia guilliermondii against grey mold of apples and its possible modes of action

The efficacy of Pichia guilliermondii strain M8 against Botrytis cinerea on apples was evaluated under storage conditions, and its possible modes of action were investigated both in vitro and in vivo experiments. After storage at 1 °C for 120 days, M8 reduced grey mold incidence from 45.3% (control) to 20.0%. In apple juice medium (AJM) and in wound-inoculated apples, M8 at 10⁹ and 10⁸ cells ml⁻¹ inhibited the spore germination of B. cinerea and the grey mold development. When co-culturing B. cinerea in vitro or in vivo in the presence of the yeast, neither inactivated cells nor culture filtrate of the yeast had any effect on spore germination or germ tube elongation. In AJM, the spore germination was significantly recovered by the addition of 1% glucose, sucrose and fructose, or 0.5% and 1% of (NH₄)₂SO₄, phenylalanine and asparagine. When the pathogen and the yeast were co-incubated in apple wounds with addition of the same nutrients, the inhibition of rots was significantly reduced by the supplemental nutrients. Light microscopy revealed that the yeast strongly adhered to the hyphae and spores of B. cinerea. M8 produced hydrolytic enzymes, including β-1,3-glucanase and chitinases in minimal salt media with different carbon sources. Pretreatment with M8 at 10⁸ cells ml⁻¹ followed by washing, significantly reduced grey mold lesions, suggesting an induction of defense responses. Direct attachment, competition for nitrogen and carbon sources, secretion of hydrolytic enzymes and induction of host resistance play a role in the biocontrol mechanism of P. guilliermondii M8 against B. cinerea.     

Screening and identification of yeast strains from fruits and vegetables: Potential for biological control of postharvest chilli anthracnose (Colletotrichum capsici)

Yeasts antagonistic to Colletotrichum capsici were isolated from Thai fruits and vegetables. Four antagonists (R13, R6, ER1, and L2) were found that inhibited C. capsici growth with biocontrol efficacies of 93.3%, 83.1%, 76.6%, and 66.4%, respectively. Identification by 26S rDNA, and ITS region sequence together with physiological and morphological characteristics, showed them to be Pichia guilliermondii, Candida musae, Issatchenkia orientalis, and Candida quercitrusa, in order of their efficacy. P. guilliermondii strain R13 showed efficacy in reducing disease incidence on C. capsici infected chilli fruits to as low as 6.5%. Lower disease incidence was observed at lower storage temperature. The application of P. guilliermondii is more effective for preserving chilli fruits than conventional preservation with chlorinated water.     

Identification and characterization of chitin synthase genes in the postharvest citrus fruit pathogen Penicillium digitatum

In this study, we carried out the isolation and characterization of chitin synthase genes (CHS) of the main citrus fruit postharvest pathogen Penicillium digitatum. Using distinct sets of degenerate primers designed from conserved regions of CHS genes of yeast and filamentous fungi, PCR methods, and a DNA genomic library, five putative CHS genes (PdigCHSI, PdigCHSII, PdigCHSIII, PdigCHSV, and PdigCHSVII) were identified, isolated, sequenced, and characterized. Phylogenetic analyses, sequence identity, and domain conservation support the annotation as CHS. A very high sequence identity and strong synteny were found with corresponding regions from the genome of Penicillium chrysogenum. Gene expression of P. digitatum CHS genes during mycelium axenic growth, under oxidative and osmotic stress conditions, and during infection of citrus fruits was confirmed and quantified using quantitative RT-PCR (qRT-PCR). PdigCHSIII had the highest expression among the five genes by one order of magnitude, while PdigCHSII had the lowest. However, PdigCHSII was strongly induced coincident with conidial production, suggesting a role in conidiogenesis. The expression of PdigCHSI, PdigCHSIII, PdigCHSV, and PdigCHSVII was upregulated during infection of citrus fruit. PdigCHSV and PdigCHSVII coexpressed in most of the experiments carried out, and they are separated by a 1.77 kb intergenic region and arranged in opposite directions.     

Screening and identification of epiphytic yeasts with potential for biological control of green mold of citrus fruits

Epiphytic yeasts isolated from the surface of citrus fruits, harvested in several orchards in the Souss-Massa-Draa Valley, Agadir, Morocco, were in vivo screened for antagonistic activity against Penicillium digitatum, the causal agent of green mold of citrus. From a total of 245 yeast strains assessed for their biocontrol activity against P. digitatum, fifteen reduced the incidence of disease to less than 50%. The effectiveness of the best selected yeast strains showed that Pichia anomala (YT73), Debaryomyces hansenii (YT22) and Hanseniaspora guilliermondii (YT13) were the most effective, with a reduction of green mold incidence from 65 to ~80%, compared to the control. The identification of the fifteen selected yeast strains was carried out through an integrated approach including phenotypic and genotypic (sequencing of D1/D2 domain of 26S rDNA encoding gene) methods. These 15 selected were identified as: H. guilliermondii, D. hansenii, H. uvarum and P. anomala. The study of the dynamics of two of the best strains, H. guilliermondii and D. hansenii, showed that these strains can grow rapidly, by approximately 2 log units, in citrus fruit wounds. Such rapid growth in wounds indicates that these antagonist yeasts are excellent colonizers of citrus wounds and can thrive on citrus fruits as a substrate.     

Biological control of crown rot of bananas with Pichia anomala strain K and Candida oleophila strain O

The antagonistic activity of two yeast strains (Pichia anomala (E.C. Hansen) Kurtzman, strain K and Candida oleophila Montrocher, strain O) against the parasitic complex responsible for banana crown rot was evaluated. The strains were applied at three different concentrations (10⁶, 10⁷, 10⁸ cfu/ml) and their efficacy tested in vivo on three separate fungi (Colletotrichum musae (Berk. & Curt.) Arx, Fusarium moniliforme Sheldon, and Cephalosporium sp.) and on a parasitic complex formed by association of these three fungi. At the concentrations used C. musae appeared to be the most pathogenic. The complex showed intermediate aggressiveness between C. musae and both other fungi.Statistically significant antagonistic effects were observed on C. musae, F. moniliforme, and the fungal complex. The highest protection level (54.4%) was observed with strain O added at 10⁸ cfu/ml on crowns previously inoculated with the fungal complex. The level was lower when the fungi were inoculated separately.Furthermore, the antagonistic effect was strongly reinforced when strain O at 10⁸ cfu/ml was applied 24 h before fungal complex inoculation (59.9%), as compared to its application 15 min (24.3%) or 3 h (27.3%) after fungal complex inoculation. Bananas showed increased susceptibility to the fungal complex from March to June, and this influenced the level of protection by yeast, which decreased over the same period. A strict negative correlation (R² = 0.83) was highlighted between susceptibility of banana to crown rot and protection provided by yeast.     

Synthesis of Drosophila melanogaster acetylcholinesterase gene using yeast preferred codons and its expression in Pichia pastoris

To improve the expression level of recombinant Drosophila melanogaster AChE (R-DmAChE) in Pichia pastoris, the cDNA of DmAChE was first optimized and synthesized based on the preferred codon usage of P. pastoris. The synthesized AChE cDNA without glycosylphosphatidylinositol (GPI) signal peptide sequence was then ligated to the P. pastoris expression vector, generating the plasmid pPIC9K/DmAChE. The linearized plasmid was homologously integrated into the genome of P. pastoris GS115 via electrotransformation. Finally seven transformants with high expression level of R-DmAChE activity were obtained. The highest production of R-DmAChE in shake-flask culture after 5-day induction by methanol was 718.50 units/mL, which was about three times higher than our previous expression level of native DmAChE gene in P. pastoris. Thus, these new strains with the ability to secret R-DmAChE in the medium could be used for production of R-DmAChE to decrease the cost of the enzyme expense for rapid detection of organophosphate and carbamate insecticide residues.     

海洋季也蒙毕赤酵母ACP基因的克隆及生物信息学分析

酰基载体蛋白是脂肪酸途径中重要的组件,能够结合脂肪酸代谢途径中各种脂肪酰基中间体,在脂肪酸代谢中是不可或缺的辅因子。以本实验室筛选保存的海洋季也蒙毕赤酵母基因组为模板,PCR扩增酰基载体蛋白基因,获得384 bp的目的片段。生物信息学分析显示,其具有完整的开放阅读框,编码127个氨基酸,有磷酸泛酰巯基乙胺结合位点,为非分泌型亲水性蛋白,不存在信号肽,存在9个潜在的磷酸化位点,二级结构和三级结构主要以α螺旋和无规则卷曲为主,与已知的酰基载体蛋白结构有很高的相似性。     

Survey of brown soft scale Coccus hesperidum L. parasitoids in southern California citrus

The parasitoid complex of brown soft scale, Coccus hesperidum L., a multivoltine soft scale, was determined in southern California citrus over the period February 2004–March 2006. The survey was conducted by placing brown soft scale-infested yucca leaves in the canopy of citrus trees and subsequently rearing individually isolated parasitized scales in the laboratory. A total of 14 species parasitized brown soft scale in the field, the most abundant ones belonging to the genus Metaphycus Mercet (75%). The most abundant parasitoid species was Metaphycus angustifrons Compere (38% parasitism), and this is a new record of establishment for this species in California. Coccophagus species accounted for only 11% parasitism. There were important spatio-temporal differences across the parasitoid complex survey locations. We also found that the five most abundant encyrtid parasitoid species showed preferences for scales of different sizes. Our results have implications for biological control of citricola scale, Coccus pseudomagnoliarum (Kuwana), an important pest of citrus in the San Joaquin Valley of central California. Notably, this species is nearly absent in southern California. Brown soft scale is considered to be an alternate host for parasitoids of citricola scale, a univoltine soft scale, at times when the latter species is unavailable for parasitism.     

Evaluation of formulations of Bacillus licheniformis for the biological control of tomato gray mold caused by Botrytis cinerea

Bacillus licheniformis N1, which has previously exhibited potential as a biological control agent, was investigated to develop a biofungicide to control the gray mold of tomato caused by Botrytis cinerea. Various formulations of B. licheniformis N1 were developed using fermentation cultures of the bacteria in Biji medium, and their ability to control gray mold on tomato plants was evaluated. The results of pot experiments led to the selection of the wettable powder formulation N1E, based on corn starch and olive oil, for evaluation of the disease control activity of this bacterium after both artificial infection of the pathogen and natural disease occurrence under production conditions. In plastic-house artificial infection experiments, a 100-fold diluted N1E treatment was found to be the optimum biofungicide spray formulation. This treatment resulted in the significant reduction of symptom development when N1E was applied before Bo. cinerea infection, but not after the infection. Both artificial infection experiments in a plastic house and natural infection experiments under production conditions revealed that the N1E significantly reduced disease severity on tomato plants and flowers. The disease control value of N1E on tomato plants was 90.5% under production conditions, as compared to the 77% conferred by a chemical fungicide, the mixture of carbendazim and diethofencarb (1:1). The prevention of flower infection by N1E resulted in increased numbers of tomato fruits on each plant. N1E treatment also had growth promotion activity, which showed the increased number of tomato fruits compared to fungicide treatment and non-treated control and the increased fruit size compared the non-treated control under production conditions. This study suggests that the corn starch-based formulation of B. licheniformis developed using liquid fermentation will be an effective tool in the biological control of tomato gray mold.     

The potential of Ulocladium botrytis for biological control of Orobanche spp.

A strain of Ulocladium botrytis isolated from diseased Orobanche crenata shoots caused disease on the parasitic weed in pathogenicity tests. The potential of the fungus to be developed as a mycoherbicide for Orobanche spp. was further investigated. Although the fungus significantly decreased O. crenata germination in vitro by 80%, it did not generally lead to a decreased number of O. crenata shoots or tubercles in inoculated root chambers or pots. However, the number of diseased or dead tubercles and underground shoots was significantly increased compared to the noninoculated treatments. Postemergence inoculation of O. crenata shoots with a conidial suspension resulted in the death of almost all inoculated plants 14 days after application under greenhouse conditions. In preliminary host-range studies, the pathogen caused disease on Orobanche cumana on sunflower whereas on Orobanche aegyptiaca shoots parasitizing tomato only minimal disease symptoms could be detected after postemergence inoculation. Based on the results of our investigations, we conclude that Ulocladium botrytis has only a limited potential to be used as a biocontrol agent against Orobanche spp.     

荧光假单胞菌ZX对采后锦橙绿霉病的防治及其抑菌机制

【目的】采后柑橘极易受指状青霉(Penicillium digitatum)侵染而发生严重的绿霉病腐烂,生物防治因具有安全、有效、环保等特点近年来备受关注。论文旨在研究荧光假单胞菌(Pseudomonas fluorescens)ZX对采后柑橘绿霉病的防治效果,揭示P.fluorescensZX对P.digitatum可能存在的作用机制。【方法】以"北碚447"锦橙果实为试材,先分别接种20μL拮抗菌培养液、滤液(培养液离心后,上清经0.22μm滤膜过滤)、菌悬液(培养液离心后,菌体用无菌水反复洗涤并用无菌水重悬)和热杀死液(培养液高温高压灭菌),2 h后接种20μL P. digitatum孢子悬浮液(1×10~4spores/m L),所有果实于20oC、90%相对湿度环境下恒温恒湿培养8 d后,测定果实的发病率和病斑直径;制备柑橘皮培养基,进行平板抑菌试验,探索P. fluorescens ZX对P. digitatum孢子发芽情况的影响;采用两板对扣法和生物熏蒸法研究P.fluorescensZX挥发性次级代谢产物的抑菌作用;利用插入式细胞培养皿等分析P.fluorescensZX和P.digitatum之间竞争的营养物质;同时,测定P.fluorescensZX的生长曲线,利用结晶紫染色法评估P. fluorescens ZX的生物膜形成能力。【结果】P. fluorescens ZX不同处理液之间对采后锦橙绿霉病的作用效果差异显著,菌悬液抑菌效果最好,经菌悬液处理的果实,发病率和病斑直径分别仅为40.83%和1.78 cm;不论是在柑橘皮固体培养基上对峙培养还是在液体培养基中混合培养,菌悬液和原液的作用效果较好,固体平板上,相对抑制率达到了35%–45%,液体培养基中,P. digitatum孢子12 h后的发芽率不超过27%;P. fluorescens ZX产生的挥发性物质具有抑菌作用,经P. fluorescensZX熏蒸处理的锦橙果实,发病率和病斑直径都显著降低;营养竞争试验结果表明,P. fluorescens ZX能更快速有效地消耗柑橘皮培养基中的营养,并和P. digitatum竞争葡萄糖、果糖、蔗糖、天冬氨酸、苏氨酸、丝氨酸、亮氨酸、精氨酸和脯氨酸等营养物质;同时,P. fluorescens ZX生命力强,培养4 h后即进入对数生长期,约24 h后形成成熟的生物膜。【结论】P. fluorescens ZX可能通过抑制P. digitatum孢子发芽、营养与空间竞争、形成生物膜、产生抑菌物质等方式抑制P.digitatum的生长繁殖,有效防治采后锦橙绿霉病。     

Biological control of Lygus lineolaris by Peristenus spp. in strawberry

Peristenus digoneutis Loan (Hymenoptera: Braconidae) was introduced to the US for biological control of the tarnished plant bug, Lygus lineolaris (Palisot de Beauvois) (Hemiptera: Miridae), and has since spread through much of the northeast. The purpose of this study was to determine if P. digoneutis and a native congener, Peristenus pallipes (Curtis), parasitize L. lineolaris in strawberry (where it is a key pest), and what factors relate to parasitism levels. During 1997–1999 we monitored parasitism on 17 strawberry farms in 14 counties in eastern and western New York State. We found that in eastern NY (where P. digoneutis has been established since the early 1990s), overall mean parasitism was 19.7% (ranging from 0 to 70%), mostly by P. digoneutis. Mean parasitism was significantly lower (12.3%, ranging from 0 to 58%) in western NY (where P. digoneutis was first recorded in 1999), and was mostly by P. pallipes. P. pallipes parasitism was significantly lower in eastern than western NY, suggesting the potential for competitive interaction with P. digoneutis. The insecticide regime of a farm was an important factor influencing parasitism rate, which was 5- to 6.5-fold higher on organic or casually sprayed farms than on intensely treated farms, though pest density under these three regimes was not significantly different. L. lineolaris density, and parasitism rate in nearby alfalfa and abandoned fields were also significant factors for parasitism in strawberry.     

Biology of Platycephala planifrons (Diptera: Chloropidae) and its potential effectiveness as biological control agent for invasive Phragmites australis in North America

Phragmites australis is a cosmopolitan clonal grass valued for its support of diversity-rich communities in its native range and feared for its devastating effects on native diversity where the species is introduced. Lack of successful control in North America resulted in the initiation of a biological control program. We used a combination of field surveys and common garden experiments in Europe to study life history and ecology of a chloropid fly, Platycephala planifrons, to assess its potential as a biological control agent. The fly is widely distributed (in non-flooded sites) throughout Eurasia but attack rates are generally low (mean 5–10%; max. 29%). Adults emerge in late June and may live for several months. Females lay eggs at the base of Ph. australis shoots. First instar larvae of this stem-feeding fly overwinter in dormant below-ground shoots of Ph. australis and rapidly complete development in early spring. Larval feeding destroys the growing meristem of the shoot causing premature wilting and 60–70% reductions in shoot biomass production. Early season attack and considerable impact suggest that Pl. planifrons could be a potent biocontrol agent, if it can escape suppression by natural enemies in the introduced range. However, the generally low attack rates in its native range and its dependence on dry sites appear to make the species a “second-choice” candidate for potential release in North America.     

牛乳铁蛋白N-叶在毕赤氏酵母中的高效表达及抑菌性

[目的]本研究将牛乳铁蛋白的N-叶(BLF-N)克隆至毕赤氏酵母菌基因组中,通过密码子优化和发酵条件优化,实现BLF-N的异源高效表达,研究重组BLF-N的抑菌功能.[方法]本文以BLF基因为模板,按照毕赤氏酵母的密码子偏好性进行密码子优化,构建重组表达载体pPIC9K-UBLF-N,电击转化到 Pichia past...     

Intra- and interspecific competition by Fopius arisanus and Diachasmimorpha tryoni (Hymenoptera: Braconidae), parasitoids of tephritid fruit flies

Fopius arisanus (Sonan) and Diachasmimorpha tryoni (Cameron) are two important solitary endoparasitoids of tephritid fruit flies. The former species attacks host eggs while the latter attacks host larvae, and both species emerge as adults from the host puparium. This study investigated intrinsic competition between these two parasitoids, as well as aspects of intraspecific competition within each species in the Mediterranean fruit fly, Ceratitis capitata (Wiedemann). Parasitization by F. arisanus resulted in direct mortality of host eggs and prolonged development of host eggs and larvae. Superparasitism by F. arisanus was uncommon when mean parasitism per host patch was <50%, but increased with rising rates of parasitism. Superparasitism by D. tryoni was more common. In superparasitized hosts, supernumerary individuals of F. arisanus were killed through physiological suppression, while supernumerary larvae of D. tryoni were killed mainly through physical attack. In multiparasitized hosts, dissections showed that 81.6% of D. tryoni eggs in the presence of F. arisanus larvae died within 3 days, indicating physiological inhibition of egg hatch. Rearing results further showed that F. arisanus won almost all competitions against D. tryoni. The ratio of D. tryoni stings to ovipositions was lower in hosts not previously parasitized by F. arisanus than in parasitized hosts, suggesting that D. tryoni can discriminate against parasitized hosts. The mechanism that F. arisanus employs to eliminate D. tryoni is similar to that it uses against all other larval fruit fly parasitoids so far reported. The results are discussed in relation to the competitive superiority of early acting species in fruit fly parasitoids, and to a possible competitive-mediated mechanism underlying host shift by D. tryoni to attack non-target flies following the successful introduction of F. arisanus in Hawaii.     

High level expression and purification of active recombinant human interleukin-8 in Pichia pastoris

Human interleukin-8 (hIL-8) is a member of interleukin family which functions as a chemotactic factor as well as an angiogenesis mediator. Previously, a study reported that hIL-8 could be purified from inclusion bodies using a prokaryotic expression system, however, the required re-naturation step limits the recovery of fully active protein. In this study, soluble recombinant hIL-8 was expressed as a secreted protein at high level in Pichia pastoris under the control of AOX1 (alcohol oxidase 1) promoter. A simple purification strategy was established to recover rhIL-8 from the fermentation supernatant. The process includes precipitation with 80% saturation ammonium sulfate and CM Sepharose ion exchange chromatography, yielding 30 mg/L purified rhIL-8 at over 95% purity. The obtained rhIL-8 displays high specific activity, stimulating the migration of mouse neutrophils at concentrations as low as 0.25 ng/mL. Our results demonstrate that P. pastoris expression system is an efficient tool for large-scale manufacture of active recombinant hIL-8 for various applications.     

The introduction and release of Chiasmia inconspicua and C. assimilis (Lepidoptera: Geometridae) for the biological control of Acacia nilotica in Australia

Two geometrid moths Chiasmia inconspicua and Chiasmia assimilis, identified as potential biological control agents for prickly acacia Acacia nilotica subsp. indica, were collected in Kenya and imported into quarantine facilities in Australia where laboratory cultures were established. Aspects of the biologies of both insects were studied and CLIMEX® models indicating the climatically favourable areas of Australia were developed. Host range tests were conducted using an approved test list of 74 plant species and no-choice tests of neonate larvae placed on both cut foliage and potted plants. C. inconspicua developed through to adult on prickly acacia and, in small numbers, Acacia pulchella. C. assimilis developed through to adult on prickly acacia and also in very small numbers on A. pulchella, A. deanei, A. decurrens, and A. mearnsii. In all experiments, the response on prickly acacia could be clearly differentiated from the responses on the non-target species. Both insects were approved for release in Australia. Over a three-year period releases were made at multiple sites in north Queensland, almost all in inland areas. There was no evidence of either insect’s establishment and both colonies were terminated. A new colony of C. assimilis was subsequently established from insects collected in South Africa and releases of C. assimilis from this new colony were made into coastal and inland infestations of prickly acacia. Establishment was rapid at one coastal site and the insect quickly spread to other infestations. Establishment at one inland area was also confirmed in early 2006. The establishment in coastal areas supported a CLIMEX model that indicated that the climate of coastal areas was more suitable than inland areas.     

Synthesis, purification and bioactivity of recombinant human activin A expressed in the yeast Pichia pastoris

The transforming growth factor-beta (TGF-β) superfamily member, activin A, plays a central role in the regulation of multiple physiological processes including cell differentiation, mitogenesis, embryogenesis, apoptosis and inflammation. In normal cells, activin A signalling is regulated to maintain cellular and tissue health and suppress tumour growth. Disruption of activin A signalling has been implicated in tumour formation and progression. Hence, the availability of activin A is an important target for the development of diagnostics and drugs for therapeutic intervention. To this end, we have expressed human activin A in Pichia pastoris, permitting its secretion into culture medium and purification as the mature homodimer. A construct was engineered encoding the monomeric precursor protein with a N-terminal FLAG affinity tag (DYKDDDDK) and a cleavage site (EKR) for Kex2p protease. Procedures for the two-step purification of human activin A by ion-exchange and anti-FLAG antibody affinity chromatography, and for the removal of the FLAG affinity tag from purified recombinant human activin A by enteropeptidase, are described. The molecular weights of the FLAG-tagged and de-tagged human activin A were confirmed by MALDI-TOF mass spectroscopy. The biological activity of these recombinant activins was assessed for their effects on modulating the secretion of Endothelin-1 (ET-1) by human umbilical vein endothelial cells (HUVECs). The recombinant human activin A containing the intact FLAG tag resulted in a reduced ET-1 secretion from HUVECs, whereas upon removal of this affinity purification tag the purified recombinant human activin A restored ET-1 secretion to levels comparable to the positive control. These results document an approach of considerable potential for the simple, large-scale expression and purification of this important human growth factor for use in diagnostic and therapeutic purposes.