Analysis of thrips (Thysanoptera: Thripidae) diversity in avocado shows the presence of a new species and limitations of using the COI gene for differentiation of two species

Thrips are important pests of avocados causing damage to the fruits, making themunsuitable for exportation. Many of them are quarantine species and their identification isnecessary. Despite their importance, not many studies describe thrips diversity on avocado,especially in Colombia, partly because identification of some of the species is difficult usingclassical taxonomic keys. In this study, 93 adult thrips specimens were collected on avocadoand adventitious dandelion plants from plantations in the department of Antioquia, Colombia. Morphological characters were used to classify 62 of them and all of them were analyzed by PCR amplification and sequencing of cytochrome c oxidase subunit I (COI) gene and internal transcribed spacer (ITS) region. The species identified by morphological features were Frankliniella gardeniae Moulton, F. gossypiana Hood, F. panamensis Hood, Liothrips perseae Watson, Thrips palmi Karny, T. simplex (Morison), T. trehernei Priesner, Microcephalothrips abdominalis (Crawford DL), and Scirtothrips hansoni Mound & Hoddle (Tubulifera), being F. panamensis, M. abdominalis, and S. hansoni reported for the first time on avocado in Colombia. Thrips simplex and T. trehernei were also found on adventitious dandelion plants, but not on avocado. Phylogenetic analysis of COI and ITS sequences showed that most species, except for F. gardeniae and F. gossypiana were mainly identified with the COI gene. Furthermore, FST values obtained between them suggest no genetic differentiation at COI level. The results obtained here contribute to the knowledge of the diversity of thrips species found on the avocado crop in Colombia and are meaningful for pest management.

Unmarked gene editing in Clavibacter michiganensis using CRISPR/Cas9 and 5-fluorocytosine counterselection

Plant pathogenic bacteria in the genus Clavibacter are important quarantine species that cause considerable economic loss worldwide. The development of effective gene editing techniques and additional selectable markers is essential to expedite gene functional analysis in this important Gram-positive genus. The current study details a highly efficient unmarked CRISPR/Cas9-mediated gene editing system in Clavibacter michiganensis (Cm), which couples the expression of cas9 and sgRNA with homology-directed repair templates and the negative selectable marker codA::upp within a single plasmid. Initial experiments indicated that CRISPR/Cas9-mediated transformation could be utilized for both site directed mutagenesis, in which an A to G point mutation was introduced at the 128th nucleotide of the Cm rpsL gene to generate a streptomycin resistant mutant, and complete gene knockout, in which the deletion of the Cm celA or katA genes resulted in transformants that lacked cellulase and catalase activity, respectively. In subsequent experiments the introduction of the codA::upp cassette into the transformation vector facilitated the counterselection of unmarked transformants by incubation in the absence of the selective antibiotic, followed by plating on M9 agar containing 100 μg/ml 5-fluorocytosine (5-FC), in which an unmarked katA mutant lacking the transformation vector was recovered. Compared to conventional homologous recombination, the unmarked CRISPR/Cas9-mediated system was more useful and convenient as it allowed the template plasmid to be reused repeatedly to facilitate the editing of multiple genes, which constitutes a major advancement that could revolutionize research into Cm and other Clavibacter species.

Weed seed contamination in imported seed lots entering New Zealand

Imports of seeds for sowing are a major pathway for the introduction of contaminant seeds, and many agricultural weeds globally naturalised originally have entered through this pathway. Effective management of this pathway is a significant means of reducing future plant introductions and helps minimise agricultural losses. Using a national border inspection database, we examined the frequency, origin and identity of contaminant seeds within seed for sowing shipments entering New Zealand between 2014–2018. Our analysis looked at 41,610 seed lots across 1,420 crop seed species from over 90 countries. Overall, contamination was rare, occurring in 1.9% of all seed lots. Among the different crop types, the arable category had the lowest percentage of seed lots contaminated (0.5%) and the forage category had the highest (12.6%). Crop seeds Capsicum, Phaseolus and Solanum had the lowest contamination rates (0.0%). Forage crops Medicago (27.3%) and Trifolium (19.8%) had the highest contamination rates. Out of 191 genera recorded as contaminants, Chenopodium was the most common. Regulated quarantine weeds were the rarest contaminant type, only occurring in 0.06% of seed lots. Sorghum halepense was the most common quarantine species and was only found in vegetable seed lots. Vegetable crop seed lots accounted for approximately half of all quarantine species detections, Raphanus sativus being the most contaminated vegetable crop. Larger seed lots were significantly more contaminated and more likely to contain a quarantine species than smaller seed lots. These findings support International Seed Testing Association rules on maximum seed lot weights. Low contamination rates suggest industry practices are effective in minimising contaminant seeds. Considering New Zealand inspects every imported seed lot, utilises a working sample size 5 times larger than International Seed Testing Association rules require, trades crop seed with approximately half of the world’s countries and imports thousands of crop seed species, our study provides a unique overview of contaminant seeds that move throughout the seed for sowing system.

Environmental niche modelling as tool for pest risk assessment

The article reviews the concept and technology of pest risk assessment for the spread of quarantine species based on ecological and geographical analysis and ecological niche modeling. Using a quarantine species Ipomoea hederacea (L.) Jacq. as an example, we calculated the ecological requirements of the species. The main ecological factors limiting the spread of I. hederacea were determined and the ecological amplitudes of the species in relation to each factor limiting its distribution were quantitatively estimated. We identified ecologically suitable habitats and compiled a map of the ecological-geographical niche of the species using specially created environmental maps based on the obtained data of the ecological limits of the species. Using the map of the ecological-geographical niche, the I. hederacea distribution risk in the regions of the Russian Federation was estimated. Similar maps can be used for basis of quantitative as well as targeted risk assessment of penetration and establishment of harmful organisms. Obtained information will be useful to compile lists of quarantine species, estimate the potential areas of geographic distribution of pests into Russian Federation, and make decisions for introduction of effective phytosanitary measures to prevent the penetration of these invasive organisms. It can also be used as the basis for organizing and conducting phytosanitary monitoring in the Russian Federation.

The stem nematode Ditylenchus dipsaci in sugar beet: a species of extremes.

The stem nematode Ditylenchus dipsaci is a migratory endoparasitic nematode of worldwide importance. D. dipsaci was long considered as a species with up to 30 different host races with specific host crop spectra. However, more recent phylogenetic studies showed that isolates from agricultural plant species, including sugar beet, should be considered as D. dipsaci sensu stricto. It is regulated as a quarantine species in many countries and classified as a regulated non-quarantine pest in the European Union to avoid further spread of this nematode by infested seeds or planting material. This chapter discusses the economic importance, distribution, host range, damage symptoms, biology and life cycle, interactions with other nematodes and pathogens, recommended integrated nematode management, and management optimization of D. dipsaci. Future research requirements and future developments are also mentioned.

Specific Identification Method based on PCR for Drosophila melanogaster

D. melanogaster is one of the most harmful citrus fruit flies having a large number of host plants. The molecular diagnostic method has been created for identification the D. melanogaster from another non-quarantine species Drosophila spp. The proposed method for differentiation is to use the mitochondrial DNA cytochrome oxidase I gene region 709-bp. We amplified samples of DNA with primers Droso-S391 and Droso-A381 by D. melanogaster, D. suzukii, and D. Simulans collections in the laboratory samples from many countries and contrasted with sequences of other GenBank Drosophila taxa. The findings of a polymerase chain reaction (PCR) based on DNA sequence polymorphisms showed that these primers accurately identify the area of the gene as well as the unique primers of Drosophila melanogaster.

The study of agricultural pests of the genus Liriomyza (Diptera: Agromyzidae) in Nagorno-Karabakh

The goal of the research is to study polyphagous species of the genus Liriomyza (Diptera, Agromyzidae) in Nagorno-Karabakh. The vegetable leaf-miner L. sativae (Blanchard, 1938), the tomato leaf-miner L. bryoniae (Kaltenbach, 1858) and the cabbage or serpentine leaf-miner L. brassicae (Riley, 1885) have been revealed among them. They affect mainly plants from the families Brassicaceae, Cucurbitaceae, Fabaceae, Solanaceae, Asteraceae. The quarantine species L. sativae has been described in the Caucasus for the first time. The paper presents distinctive features of the genus Liriomyza, a morpho-biological description of the imagoes, a structure of their wings, male genitalia, in particular the aedeagus, structural features of larvae and pupae of certain species, mines formed by the feeding of larvae, their harmfulness on the leaves of various plants. In terms of the number of host plant species, as well as the extensiveness and intensity of plant infection, the most common pest is L. sativae, the least common and harmful is L. brassicae. The larvae of L. sativae often mine the leaves of Phaseolus vulgaris and Solanum lycopersicum. Methods of a variance analysis have revealed that the sexual dimorphism of the studied species is most expressed in L. bryoniae.

THE POTENCY OF ANGLE MEASUREMENT AND COMPARISON OF VEIN LENGTHS IN DISTINGUISHING BACTROCERA SPECIES COMPLEXES

The potency of angle measurement and comparison of vein lengths in distinguishing Bactrocera species complexes. One of the most conspicuous features of Bactrocera fruit flies is their wing, which can be elaborated for identification. The distinctive wing patterns are used to separate species and classify species complexes. The wing shape can be used as a potential discriminator between closely related taxa. To develop wing applications in taxonomy, in this study, the degree measure of angles and the comparisons of vein lengths were investigated quantitatively to distinguish among important pest quarantine species of the Bactrocera dorsalis complex: B. dorsalis and B. carambolae; the B. frauenfeldi complex: B. frauenfeldi and B. albistrigata; and a species belonging to subgenus Zeugodacus, B. cucurbitae. The result showed that species complexes were clustered into significantly different groups based on the degree measure of cell br and the comparison between r-m and dm-cu cross-vein length. This conclusive evidence was supported by phylogenetic analysis using COI gene. This present result indicated that the cell br angle measure and the comparison between r-m and dm-cu vein length could be applied to distinguish species complexes in genus Bactrocera.

EXTENSION AND HARMFULNESS OF CALIFORNIAN SHIELD IN APPLE ORCHARDS

A dangerous pest is widespread in Uzbekistan, one of which is the dangerous coccidus. These include quarantine species that are considered dangerous in the republic's horticulture, including the California scale insect. In the course of observations (Tashkent region, 2018-2019) apple varieties are imported from Europe “Renet Simirenko” 6%, “White Rosemary” 8%, winter “Yellow Parmen” 5% and North American varieties “Yellow Belfleur” 4%, “Vinesep”3%, “Janatan”4%, “Jonored”5%, “Golden Delishes”8%, “King David”7%, local varieties “Samarkand First”16%, “Tashkent Borovinkasi”15% and “Nafis”19% were damaged by the California scale.