Phenotypic and genetic characterization of Pseudomonas syringae strains associated with the recent citrus bacterial blast and bacterial black pit epidemics in Tunisia

In the spring of 2012, symptoms of a disease resembling citrus blast and citrus black pit were observed in some orchards in Tunisia. The epidemic spread rapidly in the following years. Twenty‐four commercial citrus orchards from four Tunisian regions showing characteristic symptoms of bacterial diseases were surveyed during a 3‐year study. Eighty‐eight Pseudomonas‐like bacterial isolates were successfully obtained from the northeast and west of Tunisia. No isolates were recovered from the central region. Overall, 46 isolates were identified as Pseudomonas syringae pv. syringae and most of them showed similar phenotypic and genetic profiles. The virulence of three selected isolates differed from one plant cultivar to another as well as from the type of plant organ used for the inoculation. In a bioassay test, all isolates produced syringomycin, which was confirmed by molecular detection based on the syrB and syrD genes. Only EC122 possessed syrD but not syrB. DNA fingerprints, based on repetitive sequence‐based polymerase chain reaction (rep‐PCR) and PCR melting profile (PCR MP), were used to determine the potential genetic diversity among strains. Clustering of PCR MP fingerprinting data matched with rep‐PCR fingerprinting data. The generated distribution tree showed that Tunisian isolates were closely related to the citrus reference strain LMG5496. In contrast, EC112, isolated from citrus, and the almond isolate EC122 were distantly related to the type strain LMG1247^T isolated from lilac. Such studies have not been reported until now for P. syringae from citrus.     

Antiphytoviral activity of bruceine-D from Brucea javanica seeds

BACKGROUND: Brucea javanica (L.) Merr. is widely distributed throughout the southern parts of China and has been used in traditional medicine to treat a variety of diseases. The objective of the present study was to identify the active antiphytoviral compound in the seeds of B. javanica and evaluate the inhibitory activity of the compound against plant virus. RESULTS: Bioassay-guided fractionation of the most active extract from the seeds led to the isolation of an antiphytoviral compound which was identified as bruceine-D by conventional spectroscopy methods. The compound exhibited significant inhibitory activity against the infection and replication of tobacco mosaic virus (TMV), with IC(50) values of 13.98 and 7.13 mg L(-1) respectively. The compound also showed a strong inhibitory effect on the infectivity of potato virus Y (PVY) and cucumber mosaic virus (CMV). Furthermore, the compound could effectively inhibit systemic TMV infection in the host tobacco plant under glasshouse conditions.CONCLUSION: The results suggested that bruceine-D from Brucea javanica may have the potential to be used as a natural viricide, or a lead compound for new viricides.     

Trends and expectations for research and technology in the Asia‐Pacific region

The present paradigm in weed science should be restructured to meet the challenges of the 21st century for sufficient food production without environment hazards. The scope of weed science should be expanded to accommodate biotechnology in weed science, to breed genetically modified (GM) crops, allelopathic (weed‐suppressing) crops and highly competitive crops, and to utilize all the useful genes for bioproduction through re‐evaluation of the virtues of weeds and weed relatives. Adoption of GM crops needs further testing for all possible risks suggested. Risk assessment and monitoring of herbicides are needed in an agro‐ecosystem. International collaboration is needed to share information on weed management practices and to install international centers for weed inventory and farmer participatory training.     

Evaluation of loop‐mediated isothermal amplification (LAMP) assays based on 5S rDNA‐IGS2 regions for detecting Meloidogyne enterolobii

A loop‐mediated isothermal amplification (LAMP) assay for detection of Meloidogyne enterolobii (Me‐LAMP) was developed based on the sequences of the 5S ribosomal DNA (5S rDNA) and intergenic spacer 2 (IGS2) segment. The LAMP amplification was achieved at 65°C isothermal conditions within 1–1·5 h. Its amplicons were confirmed using gel electrophoresis, SacI enzyme analysis, lateral flow dipstick (LFD) assay, and visual inspection through SYBR Green I and calcein staining. The results demonstrated that the Me‐LAMP was able to specifically detect M. enterolobii populations from different geographical origins, with a detection limit of about 10 fg M. enterolobii genomic DNA, which was 10–100 times more sensitive than conventional PCR. In addition, the applicability of LAMP to field detection was confirmed following its successful performance in detecting the pest on root and soil samples. The Me‐LAMP assay possessed the characteristics of simplicity, sensitivity and specificity, and is a promising and practical molecular tool for M. enterolobii diagnosis in pest quarantine and field surveys.     

The role of altered acetyl-CoA carboxylase in conferring resistance to fenoxaprop-P-ethyl in Chinese sprangletop (Leptochloa chinensis (L.) Nees)

From paddy field observations in 2002 and 2004, fenoxaprop-P-ethyl resistance in Chinese sprangletop (Leptochloa chinensis (L.) Nees) has been studied using information collected from 11 sites in the Saphan-Sung district of Bangkok, Thailand. The resistant Chinese sprangletop was found in nine rice fields, whereas the susceptible Chinese sprangletop was found in only two rice fields. In greenhouse experiments, both fenoxaprop-P-ethyl-resistant and susceptible Chinese sprangletop from the same location were investigated for 50% growth reduction based on phytotoxicity, plant height and fresh and dry weight. The resistant Chinese sprangletop showed apparent resistance at 14-21 days after herbicide application at a rate of 21.1-337.6 g AI ha(-1). The resistance index of resistant Chinese sprangletop was 10-25 times higher than that of the susceptible Chinese sprangletop. In addition, Chinese sprangletop did not exhibit multiple resistance to oxadiazon, propanil and quinclorac. According to acetyl-CoA carboxylase (ACCase) assays, the level of ACCase specific activity in the resistant Chinese sprangletop was significantly higher than that in the susceptible Chinese sprangletop. Similarly, the ACCase activity of the resistant Chinese sprangletop was 10 times less sensitive to fenoxaprop-P-ethyl than that of the susceptible Chinese sprangletop, based on the I50 values. The present study of the mechanism responsible for resistance in the biotypes investigated indicated that there was a close association between the concentration-response at the whole-plant level and ACCase sensitivity to fenoxaprop-P-ethyl, and resistance to fenoxaprop-P-ethyl was conferred by a modified ACCase at the target site, as suggested by higher specific activity and less sensitivity to the herbicide.     

A novel amino acid substitution Ala-122-Tyr in ALS confers high-level and broad resistance across ALS-inhibiting herbicides

BACKGROUND: Wild radish, a problem weed worldwide, is a severe dicotyledonous weed in crops. In Australia, sustained reliance on ALS‐inhibiting herbicides to control this species has led to the evolution of many resistant populations endowed by any of several ALS mutations. The molecular basis of ALS‐inhibiting herbicide resistance in a novel resistant population was studied. RESULTS: ALS gene sequencing revealed a previously unreported substitution of Tyr for Ala at amino acid position 122 in resistant individuals of a wild radish population (WARR30). A purified subpopulation individually homozygous for the Ala‐122‐Tyr mutation was generated and characterised in terms of its response to the different chemical classes of ALS‐inhibiting herbicides. Whole‐plant dose‐response studies showed that the purified subpopulation was highly resistant to chlorsulfuron, metosulam and imazamox, with LD₅₀ or GR₅₀ R/S ratio of > 1024, > 512 and > 137 respectively. The resistance to imazypyr was found to be relatively moderate (but still substantial), with LD₅₀ and GR₅₀ R/S ratios of > 16 and > 7.8 respectively. In vitro ALS activity assays showed that Ala‐122‐Tyr ALS was highly resistant to all tested ALS‐inhibiting herbicides. CONCLUSION: The molecular basis of ALS‐inhibiting herbicide resistance in wild radish population WARR30 was identified to be due to an Ala‐122‐Tyr mutation in the ALS gene. This is the first report of an amino acid substitution at Ala‐122 in the plant ALS that confers high‐level and broad‐spectrum resistance to ALS‐inhibiting herbicides, a remarkable contrast to the known mutation Ala‐122‐Thr endowing resistance to imidazolinone herbicide. Copyright © 2012 Society of Chemical Industry     

Characterization and PCR-based detection of benzimidazole-resistant isolates of Monilinia laxa in California

Monilinia laxa is a pathogen of brown rot of stone fruit and almond in California, causing blossom blights and fruit rots. In this study, low-level resistance to the benzimidazole fungicides benomyl and thiophanate-methyl was detected in field isolates of M laxa collected from stone fruits and almonds in California. Low-resistant (LR) isolates grew in potato dextrose agar (PDA) plates amended with benomyl and thiophanate-methyl at 1 and 5 microg ml(-1), respectively, but not in plates amended with benomyl at 5 microg ml(-1) or thiophanate-methyl at 50 microg ml(-1). The benzimidazole LR isolates were characterized by temperature sensitivity and the DNA sequence of the beta-tubulin gene. The LR isolates showed high-temperature sensitivity, being sensitive to 1 microg ml(-1) of benomyl at 28 degrees C but resistant at 8-24 degrees C. Analysis of the DNA sequence of the beta-tubulin gene showed that the LR isolates had a point mutation at the amino-acid position 240, causing substitution of leucine by phenylalanine. Based on the point mutation, a pair of allele-specific PCR primers was developed for rapid detection of LR isolates of M laxa. In addition, a pair of PCR primers specific to M laxa was developed on the basis of the differences in the DNA sequence of the intron 6 of beta-tubulin gene from M laxa, M fructicola and other fungal species. The primer pair amplified the expected 376-bp DNA fragment from all M laxa isolates tested, but not from 14 other fungal species isolated from stone fruit and almond crops. The restriction endonuclease BsmA I recognized the sequence GTCTCC in the PCR products from sensitive (S) isolates only, but not the GTTTCC sequence in the PCR products from LR isolates. The endonuclease digested the 376-bp PCR products from S isolates to produce two bands (111 and 265 bp) on agarose gels. Thus, both allele-specific PCR and the PCR-restriction fragment length polymorphism (PCR-RFLP) methods could be useful for rapidly detecting benzimidazole-resistant isolates of M laxa from stone fruit and almond crops in California.     

昆虫病毒在害虫防治上的应用及其对寄生蜂的影响

本文综述了昆虫病毒，包括核多角体病毒（NPV）、昆虫痘病毒（EPV）和颗粒体病毒（GV），在防治农林害虫中的应用及其对寄主寄生蜂影响的研究进展，同时也介绍了昆虫杆状病毒诱导细胞凋亡及基因工程研究的近况。