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The leaf miner Coptodisca lucifluella and the carpophagous Rhagoletis completa, of American origin, are two non‐native walnut (Juglans spp.) pests in Italy. For the Friuli Venezia Giulia region (FVG) (N‐E Italy), C. lucifluella is not yet listed, while R. completa has been present for over 25 years. During 2015, samples of leaves and fruits were collected from 219 single old common walnut trees (Juglans regia) placed at different altitudes (0–1,073 m a.s.l.) in FVG to detect the distribution and abundance of both pests. Samples of leaf miner larvae and their parasitoids were subjected to mitochondrial DNA analysis for identification. C. lucifluella has been found in 55 out of 219 sites. The species has been identified by typical leaf symptoms and by its DNA barcode. This is the first report of the species for FVG. The leaf miner distribution was negatively correlated with altitude. The species has not been observed at sites over 600 m a.s.l. Larvae were parasitized by native parasitoids identified as belonging to the genus Chrysocharis by morphological features and by DNA barcode. R. completa has been found in 89 out of 165 sites on walnut trees with fruits. The infestation level was very high in lowland localities. At sites over 700 m a.s.l., no infestation was found. Both species are widespread in the region, and their occurrence is significantly affected by altitude. Data suggest that walnut trees could be cultivated in some mountain areas without the need to control R. completa with insecticides.

Background: Walnut oil use is currently limited by its poor oxidative stability due to the high percentage of polyunsaturated fatty acids. Modifying the oil composition may be a goal in walnut breeding to increase interest in this crop. Exploring natural variability and identifying the main environmental factors affecting oil quality are necessary in crop selection. Therefore 190 wild accessions were collected and evaluated during 2013 and 2014 for oil content and its fatty acid profile and compared with five commercial cultivars as references.; Results: High variation in kernel oil content and fatty acid composition was found in the native walnut. Kernel oil content ranged from 54.2 to 72.2% (w/w). The major fatty acids were linoleic (range 46.9-68.6%), oleic (10.0-25.1%), linolenic (6.9-17.6%), palmitic (3.9-11.4%) and stearic (1.1-5.2%) acids. Some accessions had oil with a fatty acid ratio very different from the reference commercial cultivars, especially the oleic acid/polyunsaturated fatty acid (PUFA) ratio. A significant linear relationship and positive correlation between the daily minimum temperature and oleic acid content was observed in the wild walnuts.; Conclusion: The wide variation in fatty acid content and composition allows superior accessions to be selected for diffusion among growers. A suitable strategy would be to make a selection against PUFA content rather than just for high oleic acid. In addition, the selected high oleic accessions, before being utilized per se or as donor parents in breeding programs, have to demonstrate they are not adversely affected by the environment. © 2017 Society of Chemical Industry.; © 2017 Society of Chemical Industry.

Walnut oil use is currently limited by its poor oxidative stability due to the high percentage of polyunsaturated fatty acids. Modifying the oil composition may be a goal in walnut breeding to increase interest in this crop. Exploring natural variability and identifying the main environmental factors affecting oil quality are necessary in crop selection. Therefore 190 wild accessions were collected and evaluated during 2013 and 2014 for oil content and its fatty acid profile and compared with five commercial cultivars as references.High variation in kernel oil content and fatty acid composition was found in the native walnut. Kernel oil content ranged from 54.2 to 72.2% (w/w). The major fatty acids were linoleic (range 46.9-68.6%), oleic (10.0-25.1%), linolenic (6.9-17.6%), palmitic (3.9-11.4%) and stearic (1.1-5.2%) acids. Some accessions had oil with a fatty acid ratio very different from the reference commercial cultivars, especially the oleic acid/polyunsaturated fatty acid (PUFA) ratio. A significant linear relationship and positive correlation between the daily minimum temperature and oleic acid content was observed in the wild walnuts.The wide variation in fatty acid content and composition allows superior accessions to be selected for diffusion among growers. A suitable strategy would be to make a selection against PUFA content rather than just for high oleic acid. In addition, the selected high oleic accessions, before being utilized per se or as donor parents in breeding programs, have to demonstrate they are not adversely affected by the environment. © 2017 Society of Chemical Industry.

Juglans regia L. is distributed primarily across temperate and subtropical regions of the Northern Hemisphere. During the last glaciation, the species survived in refugial areas that in Europe included the Balkans and the Italian peninsula, two areas joined by a corridor represented by the Friuli Venezia Giulia region, where two germplasm reservoirs met and likely intercrossed during re-colonization after the last glaciation. In this work, two hundred and fifteen wild accessions native to the area were sampled, georeferenced, and genotyped with 20 microsatellite loci selected from the literature. The local accessions of this study displayed moderate genetic diversity with 80 alleles identified. The number of alleles/loci was 4.0 (4.7 alleles for the genomic SSRs (Simple Sequence Repeats) and 2.7 alleles per EST (Expressed Sequence Tag)-derived SSR, on average). An analysis of molecular variance (AMOVA) revealed that most of the molecular diversity was between individuals (nearly 98% of variation explained). The model-based clustering algorithms implemented either in STRUCTURE and GENELAND software revealed two clusters: The first one encompassed most of the samples and showed a great genetic admixture throughout the five sampling areas defined on the base of orographic characteristics of the region. The second cluster represented a small island with three samples traced back to an introduction from Russia at the beginning of the 20th century.

Few biochemical and molecular details are available on microspore growth and development. In this work, a nuclease was partially purified from diffusates of barley (Hordeum vulgare L.) microspores by using concanavalin-A as ligand. The chromatographic preparation contained a 34-kDa protein with nucleolytic activity; the enzyme (called BMN: barley microspore nuclease) was very stable at pH8.0 and temperatures below 50 degrees C. Activity was highest at pH 5.6 and increased almost exponentially with temperature until a breakpoint between activity and stability was reached at 70 degrees C. Although BMN was able to cleave RNA, the enzyme showed a remarkable preference for DNA, especially in the single-stranded form. The best homopolymeric substrates were poly(dA) and poly(A), whereas poly(dC), poly(G) and poly(I) were almost completely uncleaved. When incubated with intact nuclei, BMN caused a nucleosomal DNA ladder of approximately 200 bp. On the basis of DNA laddering, substrate specificity, Mg2+ -dependence and best performance at apoplastic pH, BMN can be referred to as a putative apoptotic nuclease involved in pollen development.

It was previously demonstrated that some plant inhibitors are able to inactivate the bacterial proteinase subtilisin ; since information concerning the effect of plant inhibitors on other microbial proteinases remains limited, we decided to determine the activity of the soybean Kunitz and Bowman-Birk inhibitors (KI and BBI, respectively) on 14 proteinases of fungal and bacterial origin. The results show that microbial proteinases are frequently inhibited by KI and BBI and that proteinases with the same EC number (eg subtilisin Carlsberg and subtilisin BPN') may equally give different responses to the inhibitors. In particular, all serine proteinases examined were affected by both KI and BBI while metalloproteinases were not. Inhibition was also achieved on a range of microbial proteinases for which the mechanistic class is yet to be established ; the data suggest that they belong to the serine type. In one instance, activation instead of inhibition was noted.

Experiments were designed to determine the seed content of protease inhibitors in different plants of a plot or at different nodes along the main stem. The inhibitory activity of the samples was measured in vitro using sodium benzoyl-DL-arginine-p-nitroanilide as chromogenic substrate and bovine trypsin as target enzyme. Six soya bean (Glycine max Merr) strains were used, three capable of synthesizing the Kunitz trypsin inhibitor (L62-364, Williams 82, and 594-1) and three lacking this particular function (L81-4590, L81-4871, and L83-4387). In all genotypes on trial, remarkable plant-to-plant variation was found, so that most strains had at least one part of the data distribution in common. Significant differences were also noted for antitryptic activities of seed samples collected at different positions on the main stem. Gradients interior to the plant varied according to the tested strain but those of Williams 82 and of its near isogenic line L81-4590 were very similar, indicating that the amount and type of variation depended on genotype and that changes in the inhibitory activity were attributable to variation in the synthesis of inhibitors other than Kunitz.

In modern, highly intensive agriculture, the control of insect pests is basically achieved with the appli- cation of chemical pesticides. Heavy reliance on this sole strategy is associated with several drawbacks, and the de- velopment of alternative or complementary methods to chemical control is desirable. In this work, three soybean genes (KTi3, C-II and PI-IV) coding for serine proteinase inhibitors were isolated by PCR and transferred to Agro- bacterium tumefaciens EHA 105, which in turn was used for transforming tobacco leaf and potato tuber discs. Bio- chemical assays confirmed that transgenic plants synthe- sized serine proteinase inhibitors; rates of expression varied among plants. The level of insect resistance (tested with Spodoptera littoralis Boisduval) was particularly high in tobacco, where many plants caused the death of all larvae. In potatoes, larval mortality was much less fre- quently achieved, but the results were still encouraging in that larval weight gain was reduced by 50% in the pres- ence of adequate amounts of inhibitor. When 8-day-old larvae were fed different KTi 3 -expressing tobacco plants, a highly significant (P

Many biochemical and molecular details are available for soybean seed proteinase inhibitors, but little is known about the quantitative regulation of structural genes. In order to fill this gap, a complete set of diallel crosses was made between inbred lines characterised by the same inhibitor array coupled with a different inhibitor content in the raw flour. The Hayman analysis revealed the presence of different regulatory elements in the parental lines, giving rise to both additive and dominance variation. Dominant alleles were found to decrease the trypsin inhibitory activity (TI activity) shown by the raw flours and appeared to have higher frequencies than recessive alleles. Significant maternal effects were also detected, particularly in crosses of low-TI activity female x high-TI activity male; maternal effects strengthened the role of the regulatory genes transferred by the female parent to the hybrid. Data fitted the simple additive-dominance model with genes independent in both action and distribution. Narrow and broad heritability values were 54% and 82% respectively, thus indicating the feasibility of lowering TI activity through selection.

Two serine-proteases (P I and P II ) were isolated from the larval midgut of Spodoptera littoralis by affinity chromatography. These hydrolysed casein and BApNA and were strongly inhibited by TLCK. On SDS-PAGE, an apparent molecular weight of 27 kDa was determined for both proteases. When mass spectrometric analysis was performed directly on the eluted fractions, one main component per peak was observed, having a molecular mass of 24,359±2 Da (P I ) and 24,448±2 Da (P II ). The enzymes had their maximum activity and stability at pH 10 and 4, respectively. P I was significantly more active than P II at 20°C or less; it was also more resistant to denaturation by heat (55 versus 50°C). A breakpoint between activity and stability was recorded at 30°C for both proteases. P II was strongly inhibited by aprotinin, leupeptin and KI, and partly inactivated by LBTI and chymostatin; P I was unaffected by LBTI and chymostatin and was also less susceptible to aprotinin and KI. On the basis of substrate degradation and inactivation rates, it was concluded that P I and P II are very different from the two proteases found in another strain of S. littoralis . Though P I and P II were found to belong to the same mechanistic class of bovine trypsin, they were not activated by Ca 2+ over the concentration range 0–20 mM. It is suggested that transgenic plants harbouring genes coding for effective P I inhibitors should have a high level of resistance towards this insect pest.