Multilocus PCR assay reveals high diversity of vegetative compatibility types in populations of Cryphonectria parasitica in Croatia

The ascomycete fungus Cryphonectria parasitica, causal agent of chestnut blight, is probably one of the best known invasive fungal pathogens in forests of Europe and North America. Mycovirus that reduces virulence of C. parasitica can be used as a biocontrol agent of the chestnut blight. However, anastomosis‐mediated virus transmission is limited by a vegetative (in)compatibility (vc) system involving at least six known diallelic vic genetic loci. This study looked at vegetative compatibility (vc) diversity in two populations of C. parasitica in Croatia. For that purpose, a PCR assay was validated and implemented using already known/published and newly designed primers for amplification of six known vic loci. The vc genotypes determined by PCR for 158 C. parasitica isolates investigated in this study were in complete agreement with the vc genotypes determined by pairwise co‐culturing of the same isolates, revealing the specificity and accuracy of the PCR‐based molecular vic genotyping assay. Twenty‐six unique vc genotypes were found among 158 isolates, and 19 vc types per population, which makes Croatian C. parasitica populations among the most diverse in Europe regarding the number of vc types and genetic diversity. Low values of multilocus linkage disequilibrium suggest sexual reproduction as a major contributor to high C. parasitica genetic diversity in studied populations.     

Effects of alternative pesticides on greenhouse whitefly in protected cultivation

The greenhouse trials were conducted to determine the efficacy of environmentally safe insecticides/fungicides with physical mode of action [Agri-50E (propylene glycol alginate, hydrated) and SB Plant Invigorator] against Trialeurodes vaporariorum (Westwood). The efficacy of Agri-50E on adults and nymphs (first, second, third, and fourth instars) was compared to the commercially available standard Boxer 200 SL (imidacloprid) and an untreated control on greenhouse-grown poinsettia. Insecticides were applied by foliar spraying, Agri-50E (2 or 3 ml/l) two times and Boxer 200 SL (1 ml/l) once. Three and 7 days after first application (DAFA), imidacloprid was more effective than both concentrations of Agri-50E in controlling T. vaporariorum adults, whereas 10 DAFA there were no differences between insecticides. Twelve DAFA, imidacloprid was more effective than Agri-50E in controlling first, second and third instars and after 19 days in controlling second, third, and fourth instars. The results obtained from the poinsettia study, indicated that Agri-50E (2 or 3 ml/l) is successful in controlling T. vaporariorum adults and moderately effective against T. vaporariorum immature stages. In the second experiment, the efficacy of Agri-50E on adults was compared to SB Plant Invigorator (SBPI), the standard treatment Calypso 480 SC (thiacloprid), and an untreated control on greenhouse-grown tomato. Agri-50E (3 ml/l) and SBPI (2 ml/l) were applied four times while Calypso 480 SC (0.4 ml/l) was applied twice. The results obtained from tomato study showed that SBPI was the most effective insecticide against T. vaporariorum adults, followed by Agri-50E and Calypso 480 EC. Beside the ecological benefits, the alternative insecticides Agri-50E and SBPI might have an important role to play in future pest management programs.     

Examining the Effects of the Destroying Ammunition,Mines, and Explosive Devices on the Presence of Heavy Metals in Soil of Open Detonation Pit: Part 1—Pseudo-total Concentration

This paper presents the results of determining the pseudo-total concentration of five heavy metals in the soil on which the destruction of ammunition, mines, and explosive devices is carried out by the method of open detonation. In the analyzed area, the concentrations of cadmium, lead, nickel, copper, and zinc were determined, while from the physical properties of the soil were determined the granulometric composition and the pH. The aim of the study is to determine the origin and total load on heavy metals and, based on that, to assess the dangers and impact of the site in terms of the soil pollution by heavy metals. In accordance with the regulations of Bosnia and Herzegovina, the results of the soil testing showed a significant load of copper (up to seven times) and cadmium (up to six times), and exceeding the allowed values for nickel and zinc in some places. Lead was the only metal whose concentration was within the maximum allowed and according to that the soil was classified as unpolluted. A sample of soil from the edge of the pit is the only sample in which all heavy metals, except Ni, were within the maximum allowable concentration. In regard to the concentration of the examined metals, the soil of the pit is classified as medium polluted from the aspect of copper, cadmium, and nickel and highly contaminated with zinc. The concentrations of copper and zinc in the examination area correspond to contaminated soil that represents ecological risk, which requires soil remediation.     

Synthetic Zeolite A as Zinc and Manganese Fertilizer in Calcareous Soil

The Leccino olive cultivar was grown in pots contained calcareous rendzina soil amended with NPK fertilizer (control treatment) or with further addition of Zeolite A (ZA) containing either zinc (Zn²⁺) or manganese (Mn²⁺) (Zn-ZA and Mn-ZA). After one year, vegetative growth and root morphological parameters were measured and elemental analysis was performed on the root, stem, and leaves. Compared to other treatments, the Zn-ZA fertilizer enhanced Zn concentration and total quantity in the root, stem, and leaves, as well as the root diameter. The root volume was greater using Zn-ZA treatment than control treatment. The total quantity of Mn in root and the root length were enhanced when using Mn-ZA compared to control or Zn-ZA treatment. According to the results of this study, it appears that zeolites containing Zn provide favorable conditions for Zn uptake in calcareous soils, while both Zn and Mn zeolites have a positive impact on olive root morphology.     

Amendment Type and Dose Effects onto Coexisting Copper,Lead, and Nickel Ions Distribution in Soil

The use of soil additives for toxic metals chemical stabilization aims to decrease in situ the pollutants’ mobility and availability. In this study, the effectiveness of rinsed red mud (RBRM) and annealed animal bones (B400) was compared in terms of Cu, Pb and Ni stabilization in two contaminated soils with contrasting properties Dystric Cambisol (CM dy) and Rendzic Leptosol (LP rz). The mobility of metals in unamended soil samples (control) and samples amended with 1% and 5% of selected additives were compared using sequential extraction protocol. The relative content of metals in readily and potentially available fractions was higher in CM dy (62% Pb, 13% Cu, and 31% Ni in exchangeable fraction) than in LP rz (<?5% of Pb, Cu, Ni in exchangeable fraction). In CM dy, both additives have caused a decrease in metal mobility with an increase of their doses. The effect of 5% sorbent addition was most pronounced related to Pb immobilization, provoking decrease of exchangeable Pb content to <?10%. Furthermore, B400 addition has redistributed investigated metals from the exchangeable to the residual phase more effectively than RBRM, and its effect on metal mobility decreased in the order Pb?>?Cu?>?Ni. Amending of LP rz soil had limited effects with no apparent decrease in exchangeable metal content. The effects of soil type variation, the type of additive and the additive dose onto metal mobility were compared according to ANOVA results. The content of readily and potentially available forms of metals was found to be (i) significantly correlated with all investigated variables for Pb, (ii) significantly correlated with soil type for Cu, and (iii) not in significant correlation with selected variables for Ni. Complex impacts of soil properties and treatment conditions on the mobility of co-contaminants emphasize the need for an individual approach to each case of contamination.     

Salinity‐induced changes in growth,superoxide dismutase activity,and ion content of two olive cultivars

Olive trees (Olea europaea L.) are considered moderately tolerant to salinity, with clear differences found among cultivars. One‐year‐old self‐rooted olive plants of the Croatian cv. Oblica and Italian cv. Leccino were grown for 90 d in nutrient solutions containing 0, 66, or 166 mM NaCl, respectively. The shoot length and the number of nodes and leaves for both cultivars were not affected by salinity up to 66 mM NaCl. However, at 166 mM NaCl, growth of Leccino was reduced earlier and to a higher extent than growth of Oblica. After 10 d of exposure to 66 and 166 mM NaCl, increased activity of superoxide dismutase (SOD) was observed in Leccino, whereas there was almost no response in Oblica. Reduced SOD activity in Leccino at 166 mM NaCl was observed after prolonged stress (90 d), whereas in Oblica SOD was increased at 66 mM compared to control or 166 mM NaCl. Electrolyte and K⁺ leakage were increased and relative water content decreased as NaCl concentration increased with similar intensity of response measured in both cultivars. Oblica exhibited an ability to keep a higher K⁺ : Na⁺ ratio at all salinity levels compared to Leccino, but since no difference was found in leaf K⁺ concentration, this was mainly achieved by less Na⁺ ions reaching the younger leaves. The antioxidative system represents a component of the complex olive salt‐tolerance mechanism, and it seems that the role of SOD in protection from oxidative stress depends on sodium accumulation in leaves.     

Diversity of vegetative compatibility types and mating types of Cryphonectria parasitica in Slovenia and occurrence of associated Cryphonectria hypovirus 1

Cryphonectria parasitica, the causal agent of chestnut blight, has been present in Slovenia since at least 1950. To improve understanding of its diversity, 254 isolates of the fungus from 11 Slovenian populations were sampled. Fifteen vegetative compatibility (vc) types were identified. The dominant vc type was EU‐13, comprising 40·1% of all isolates tested, followed by EU‐1 (19·7%), EU‐2 (12·2%) and EU‐12 (9%). The vc type diversity in the most diverse population sampled in Slovenia was higher than in the populations found previously in northern Italy and Croatia. Both mating types and perithecia were observed in surveyed populations. Natural hypovirulence was found in six out of seven populations tested, with frequencies ranging from 72·2% in the population sampled near the Croatian border to 11·1% in the population sampled near the Austrian border. All identified hypoviral isolates (21) belonged to the Italian subtype of Cryphonectria hypovirus 1 and were closely related to the hypoviruses found in other European countries. Despite the high vc type diversity, incidence of hypovirulence was also high, indicating widespread natural biological control of the disease.     

Diversity of Cryphonectria parasitica in callused chestnut blight cankers on European and American chestnut

Infection of American and European chestnuts with the chestnut blight fungus Cryphonectria parasitica results in the formation of cankers, lesions caused by the growth of mycelia within bark tissue of the host plant. Infection of the fungus with Cryphonectria hypovirus 1 (CHV‐1) results in conversion of the mycelial phenotype from virulent to hypovirulent, thus allowing production of callus around cankers as a reaction by infected trees, rendering active into inactive cankers. In this study, we sampled one USA and six European chestnut stands and assessed frequency of hypovirulent C. parasitica and diversity of vegetative compatibility (vc) types present in calluses and randomly sampled cankers. Callused cankers on C. dentata at West Salem in the USA yielded significantly more hypovirulent C. parasitica isolates compared with four sampled populations on C. sativa, while all six sampled European populations did not show any statistically significant differences among themselves. We observed no correlation between hypovirulence frequencies in randomly sampled cankers and calluses, as well as no correlation of C. parasitica vc type diversity in calluses and residential populations of the fungus. Furthermore, even though we have observed calluses with more than one vc type, they do not occur regularly. Even when present in C. parasitica populations with high vc type diversity, no more than three different vc types were observed in a single callus.     

Growth,biomass allocation and leaf ion concentration of seven olive (Olea europaea L.) cultivars under increased salinity

Olive (Olea europaea L.) is the major fruit tree in the Mediterranean region, often grown in locations where plants are exposed to increased salinity. To determine the effect of NaCl on shoot and root growth, dry matter allocation, leaf Na⁺ and K⁺ concentration, electrolyte (EL) and K⁺ leakage (KL), seven olive cultivars of different origins were grown in nutrient solution containing 0, 33, 66, 100 or 166 mM NaCl for three months. The general effect of salinity was linear and quadratic decrease of observed plant growth parameters. Different responses of tested cultivars to applied levels of salinity were found for stem dry weight, shoot length and number of leaves. As salinity increased, growth of ‘Manzanillo’ declined sharply, whereas ‘Frantoio’ was the most tolerant to growth reduction in most of the observed growth parameters. Allometric analysis showed that biomass allocation under salinity stress was similar in all cultivars, but the slope between shoot weight and total plant weight decreased as salinity increased. Since the higher allocation in roots was not found, it seems that salinity only slowed the above ground plant canopy growth. Sodium concentration in leaves of all cultivars increased as salinity increased with the highest increment reached when the salinity of nutrient solution was raised from 100 to 166 mM NaCl. Significant differences among genotypes were found in leaf Na⁺ and K⁺ concentration and K⁺:Na⁺ ratio, but they were not related to the growth rate. Generally, ‘Frantoio’ and ‘Oblica’ accumulated less Na⁺ and were able to maintain higher K⁺:Na⁺ ratios as compared to other genotypes. Electrolyte leakage and KL linearly increased with increasing salinity and the magnitude of the response depended upon the olive cultivar.