Several non-parasitic diseases are of primary significance for olive trees growing in the Mediterranean area and other warm-temperate regions of the world. As for other crops, they are generally poorly understood and often completely unrecognized, especially in olive which is considered a crop of marginal lands. The origin may rest with a lack/excess of essential nutrients or an excess of non-essential elements; it may be an unsatisfactory environment: too cold or hot, too wet or dry, or too windy; there may be unsuitable soil characteristics such as poor physical condition, water-logging, salinity, improper pH, and so on. Also pollution in the environment, spray and fire damage, and climatic extremes like lightning, hail, and snow can cause heavy losses to olive. This review focuses on the most important disorders of olive caused by environmental, physical, and chemical stresses that can affect the normal physiological processes in trees. An integrated view is utilized to highlight interactions with plant healthiness, production, and quality.

The role of some salts applied in combination with wax on the development of postharvest rots was examined on 'Tarocco' and 'Valencia late' oranges and 'Comune' Clementines. Sodium carbonate and bicarbonate, potassium carbonate and bicarbonate, ammonium bicarbonate, and potassium sorbate, at 6% concentration (w/v), in combination with a commercial wax, were evaluated for their activity against naturally occurring postharvest decay. Fruit were stored for one month at 4°C ('Tarocco' and 'Valencia late' oranges) or 6°C ('Comune' Clementine), followed by one week of shelf life at 20 ± 2°C and high RH.Most decay was from green and blue moulds, caused by Penicillium digitatum and P. italicum, respectively, with an average incidence of 11% for 'Comune' Clementines and 5% for both 'Tarocco' and 'Valencia late' oranges. Decay caused by Botrytis cinerea and Alternaria spp. was also observed. The incidence of postharvest rots on fruit treated with wax alone (11%) was higher than on those treated with water (7%), whereas in fruit treated with wax combined with different salts, decay incidence was significantly lower than with wax and water controls. In particular, potassium sorbate incorporated in wax significantly reduced the incidence of postharvest decay in all tested cultivars. The incidence of decay on fruit treated with imazalil was low, not exceeding 1%. Salts, except ammonium bicarbonate, interfered with the action of the wax to retard weight loss. The results indicate that the addition of the salts to wax may be an easy and effective mode of their application, since no additional equipment is needed.

The use of plant extracts could be a useful alternative to synthetic fungicides in the management of rot fungi during postharvest handling of fruit and vegetables. The aim of this study was to assess the in vitro and in vivo activity of extracts obtained from nine wild edible herbaceous species (Borago officinalis, Orobanche crenata, Plantago coronopus, P. lanceolata, Sanguisorba minor, Silene vulgaris, Sonchus asper, Sonchus oleraceus, and Taraxacum officinale) against some important postharvest pathogens, i.e. Botrytis cinerea, Monilinia taxa, Penicillium digitatum, P. expansum, P. italicum, Aspergillus carbonarius, and A. niger. Phenolic composition of all extracts was evaluated by HPLC. Several derivatives of caffeic acid, of the flavones apigenin and luteolin, and of the flavonols kaempferol and quercetin, were identified. Extracts from S. minor and O. crenata showed the highest efficacy in all the trials. In particular, S. minor completely inhibited in vitro the conidial germination of M. laxa, P. digitatum, P. italicum, and A. niger and strongly reduced those of B. cinerea; O. crenata extract showed a lower but still significant reduction of conidial germination on all the tested fungi. Moreover, the extracts from both species were effective in reducing the germ tube elongation also when a slight inhibition of conidial germination was observed. In many cases, a dose effect was observed, with an increase of antifungal activity as the phenolic concentration increased. In trials performed on wounded fruit, S. minor extract completely inhibited brown rot on apricots and nectarines; O. crenata extract strongly reduced grey mould, brown rot, and green mould on table grapes, apricots and nectarines, and oranges, respectively. The inhibition efficacy of extracts was ascribed to the presence of some caffeic acid derivatives and/or flavonoids. HPLC phenolic analyses provided useful information to identify the possible active compounds. (C) 2011 Elsevier By. All rights reserved.

In previous trials the flavonoid quercetin proved to be effective in reducing Penicillium expansum infections and patulin accumulation in apples. Since quercetin resulted more effective in in vivo than in in vitro trials, a possible role of this substance in enhancing host resistance was hypothesized. To verify this hypothesis, a cDNA library of genes differentially expressed in response to quercetin application was constructed by using the suppression subtractive hybridization (SSH) approach. A total of 89 unique sequences were obtained. By homology search and functional analysis the identified sequences were putatively categorized as belonging to “metabolism”, “subcellular localization” and “protein with binding functions or cofactor requirement” classes. Similarity was also found with genes coding proteins whose role in defence mechanisms is still unknown.

The effectiveness of sodium bicarbonate (SB), sodium carbonate (SC), sodium silicate (SS), potassium bicarbonate (PB), potassium carbonate (PC), potassium sorbate (PS), calcium chloride (CC), and calcium chelate (CCh) against naturally occurring postharvest decay on 'Comune' clementine and 'Valencia late' orange fruit was investigated. Aqueous salt solutions (2%, w/v, 20hlha -1) were applied according to three strategies: (i) by spraying before harvest, (ii) by dipping after harvest, and (iii) by the combination of pre- and postharvest applications. Decay was assessed after two months at 4±1°C (oranges) or 6±1°C (clementines) and 95-98% RH, followed by 7 days of shelf life at 20±2°C. For both species, preharvest sprays and the combination of pre- and postharvest applications were more effective in suppressing decay than postharvest dipping. With regard to preharvest application, several salts completely inhibited the incidence of decay as compared to the water control, namely, SC and PC on both species, and SS on 'Valencia late' oranges. In combined applications, all salts were effective in reducing the decay as compared to the water control with an efficacy varying between 66-100 and 78-100% for oranges and clementines, respectively. When salts were applied after harvest, the activity was in general less pronounced, SC and PC being the most effective on both species. In in vitro tests, the minimum inhibitory concentration (MIC) for both Penicillium digitatum and P. italicum, was achieved at 0.25% SB, SC, PB, PC, PS, and SS. The filamentous fungal population on fruit treated once in the field and with the double treatment was reduced as compared to the water control, whereas no statistical differences were observed for postharvest application. Based on these results, field application of salts can be considered a useful strategy to be included in an integrated approach for controlling postharvest diseases of citrus fruit.

Two organic media in comparison with the conventional medium, which were set up at MAIB, were tested alone or in combination with two commercial bioproducts. The media composition differentiates for the type and quantity of peat moss/compost and fertilizers. The micro and macro nutrients of the conventional medium were replaced in the two organic media by 1% (v/v) of a bio-fertilizer obtained from poultry manure. In the organic IAMB-ECOS medium, 30% of peat moss was replaced by a commercial certified compost. Trials with these media treated with a suspension of the bioproducts were carried out on grafted olive plantlets (‘Leccino’). Before transplanting, olive plantlets were inoculated by dipping roots in a conidial suspension of Verticillium dahlia. Uninoculated plantlets were used as a control. The effects of the different media, alone or in combination with bioproducts, on vegetative parameters of olive plantlets (plant height and root weight) as well as on V. dahlia infection were evaluated. Olive plantlets grown in Verticillium free media showed a significant increase in all vegetative parameters. Mainly in the organic IAMB media. Moreover, plantlets growing in the organic IAMB-ECOS, in combination with the commercial bioproducts, showed the best vegetative parameters, as well as a significant reduction of V. dahlia infection.

Within an integrated disease control strategy to promote long-term protection against Verticillium wilt of olive (Olea europaea L.), we aimed to elucidate the genetic and molecular processes taking place during the colonization of olive roots by Pseudomonas fluorescens PICF7, an effective biocontrol agent against VWO. SSH methodology and transcriptome analysis enabled the identification of putative genes differentially expressed such as several enzymes related to biosynthesis of plant hormones (i.e. lipoxygenase, chorismate synthase) and of phenylpropanoids compounds (i.e. phenylalanine ammonia lyase, cinnamyl-alcohol dehydrogenase). Interestingly, among the transcriptionally up- or down-regulated genes, diverse transcription factors implicated in plant signaling pathways for both biotic and abiotic stimulus response were also identified.

RNA interference (RNAi) is a powerful approach for elucidating gene functions in a variety of organisms, including phytopathogenic fungi. In such fungi, RNAi has been induced by expressing hairpin RNAs delivered through plasmids, sequences integrated in fungal or plant genomes, or by RNAi generated in planta by a plant virus infection. All these approaches have some drawbacks ranging from instability of hairpin constructs in fungal cells to difficulties in preparing and handling transgenic plants to silence homologous sequences in fungi grown on these plants. Here we show that RNAi can be expressed in the phytopathogenic fungus Colletotrichum acutatum (strain C71) by virus-induced gene silencing (VIGS) without a plant intermediate, but by using the direct infection of a recombinant virus vector based on the plant virus, tobacco mosaic virus (TMV). We provide evidence that a wild-type isolate of TMV is able to enter C71 cells grown in liquid medium, replicate, and persist therein. With a similar approach, a recombinant TMV vector carrying a gene for the ectopic expression of the green fluorescent protein (GFP) induced the stable silencing of the GFP in the C. acutatum transformant line 10 expressing GFP derived from C71. The TMV-based vector also enabled C. acutatum to transiently express exogenous GFP up to six subcultures and for at least 2 mo after infection, without the need to develop transformation technology. With these characteristics, we anticipate this approach will find wider application as a tool in functional genomics of filamentous fungi.

Knowledge on the genetic basis underlying interactions between beneficial bacteria and woody plants is still very limited, and totally absent in the case of olive. We aimed to elucidate genetic responses taking place during the colonization of olive roots by the native endophyte Pseudomonas fluorescens PICF7, an effective biocontrol agent against Verticillium wilt of olive. Roots of olive plants grown under non-gnotobiotic conditions were collected at different time points after PICF7 inoculation. A Suppression Subtractive Hybridization cDNA library enriched in induced genes was generated. Quantitative real time PCR (qRT-PCR) analysis validated the induction of selected olive genes. Computational analysis of 445 olive ESTs showed that plant defence and response to different stresses represented nearly 45% of genes induced in PICF7-colonized olive roots. Moreover, quantitative real-time PCR (qRT-PCR) analysis confirmed induction of lipoxygenase, phenylpropanoid, terpenoids and plant hormones biosynthesis transcripts. Different classes of transcription factors (i.e., bHLH, WRKYs, GRAS1) were also induced. This work highlights for the first time the ability of an endophytic Pseudomonas spp. strain to mount a wide array of defence responses in an economically-relevant woody crop such as olive, helping to explain its biocontrol activity.

The spread of aggressive fungal pathogens into previously non-endemic regions is a major threat to plant health and food security. Analyses of the spatial and genetic structure of plant pathogens offer valuable insights into their origin, dispersal mechanisms and evolution, and have been useful to develop successful disease management strategies. Here, we elucidated the genetic diversity, population structure and demographic history of worldwide invasion of the ascomycete Verticillium dahliae, a soil-borne pathogen, using a global collection of 1100 isolates from multiple plant hosts and countries. Seven well-differentiated genetic clusters were revealed through discriminant analysis of principal components (DAPC), but no strong associations between these clusters and host/geographic origin of isolates were found. Analyses of clonal evolutionary relationships among multilocus genotypes with the eBURST algorithm and analyses of genetic distances revealed that genetic clusters represented several ancient evolutionary lineages with broad geographic distribution and wide host range. Comparison of different scenarios of demographic history using approximate Bayesian computations revealed the branching order among the different genetic clusters and lineages. The different lineages may represent incipient species, and this raises questions with respect to their evolutionary origin and the factors allowing their maintenance in the same areas and same hosts without evidence of admixture between them. Based on the above findings and the biology of V. dahliae, we conclude that anthropogenic movement has played an important role in spreading V. dahliae lineages. Our findings have implications for the development of management strategies such as quarantine measures and crop resistance breeding.

In the framework of a project aiming to phytoremediate heavy metal contaminated soils in the Apulia region, Southern Italy, a series of greenhouse experiments followed by field trials were performed in order to optimize heavy metal phytoextraction by Brassica napus. The effects of root colonization by Bacillus licheniformis BLMB1 and of addition of municipal solid waste (MSW) composts on the capacity of B. napus to tolerate and accumulate Cr, Cu, Pb and Zn were evaluated. B. napus was able to accumulate high amount of metals in greenhouse conditions, whereas it grew with difficulty or not at all in the open field, and metal accumulation in plant fractions was relatively low. The accumulation of metals in the plant fractions was in the order: Cr>Zn>Cu>Pb. The presence of either compost or B. licheniformis BLMB1 strain enhanced metal accumulation, Cr in particular, in the experimental conditions used. This effect can be useful in the phytoextraction of Cr from contaminated soils. (C) 2010 Elsevier B.V. All rights reserved.

Greenhouse and field studies were performed to examine the growth responses and possible phytoremediation capacity towards heavy metals of several Brassicaceae (Brassica alba, Brassica carinata, Brassica napus and Brassica nigra) and Poaceae (durum wheat and barley). Soils used featured total concentrations of Cr, Cu, Pb and Zn largely exceeding the maximum levels permitted by the Italian laws. Different organic amendments were tested such as a compost and the plant growth-promoting rhizobacterium Bacillus licheniformis. In the greenhouse experiment, plant length, leaf area index and shoots dry matter were evaluated periodically for the Brassicaceae examined. Whereas plant length, grains production, weight of 1,000 seeds, ear fertility and tiller density were determined under field conditions at the end of the crop cycle for wheat and barley. In general, the species tested appeared to be tolerant to high heavy metal concentrations in soil, and slightly significant differences were found for all parameters considered. A marked growth increase was shown to occur for Brassicaceae cultivated on compost- and bacillus-amended contaminated soils, with respect to non-amended contaminated soils. With some exception, higher growth parameters were measured for wheat and barley plants cropped from contaminated soils in comparison to non-contaminated soils. Further, bacillus amendment enhanced the length of wheat and barley plants in both non-contaminated and contaminated soils, while different effects were observed for the other parameters evaluated.

In the framework of a phytoremediation project in the Apulia region (Italy) a field experiment was carried out in multi-metal contaminated soils. The accumulation and distribution of metals in different plant parts of durum wheat and barley were studied. Further, the application of Bacillus licheniformis strain BLMB1 to soil was evaluated as a means to enhance metal accumulation in plants. The translocation and the bioconcentration factors indicated that wheat and barley do not act as metal accumulators in the field conditions tested, thus phytoextraction by these species would not be recommended as a soil remediation alternative. Application of B. licheniformis improved the accumulation of all metals in roots of wheat and barley, and increased Cd, Cr, and Pb contents in the shoots of barley. Low health risk for humans and animals was evaluated to exist if straw and grain from both cereal crops grown in these contaminated sites are consumed.

The isolation in pure culture of the Xylella fastidiosa strain associated with the quick decline syndrome of olive, recently observed in Apulia (Salento peninsula, southern Italy) was attempted from symptomatic, naturally infected olive and oleander plants, and a periwinkle seedling that had been exposed to, and was infected by Xylella-positive spittlebugs. Prior to isolation, the presence of Xylella was ascertained in all donor hosts by PCR, indirect immunofluorescence and electron microscopy. solations from olive failed because of the heavy contamination by bacteria other than Xylella. By contrast, pure bacterial cultures were obtained from oleander and periwinkle extracts plated in periwinkle wilt gelrite (PWG) and buffered cysteine-yeast extract (BCYE) media. In both media, colonies were slow-growing, small-sized (less than 1 mm 25 days from plating), non pigmented, opalescent and exhibited the same morphology, except for the margin that was entire in BCYE and somewhat irregular in PWG. Bacterial cells were rod-shaped with rounded ends, had a thick and rippled cell wall, an average width of 0.35 μm, and a maximum length of ca. 5 μm. They gave a positive reaction in immunofluorence assays and were clearly decorated by colloidal gold in immunogold labelling tests. Sequenced PCR products amplified from periwinkle and oleander colonies shared 97-99% sequence identity with known X. fastidiosa strains from database and were 100% identical to one another and to comparable sequences obtained from infected olive trees. These sequences grouped in a distinct cluster of a branch comprising X. fastidiosa isolates belonging to the subspecies pauca.

The severe outbreaks of olive anthracnose observed in Apulia during the last years, caused by the occurrence of climatic conditions conducive to the disease, the reduction of cultural interventions, and the new features about the aetiology and the disease cycle, have been investigated, focusing on some epidemiological and control aspects. In particular, data on the inoculum density of Colletotrichum spp. and the incidence of latent infection on drupes have been collected. In addition, field trials on the efficacy of new QoI-based fungicides applied at different timing (new shoot growth, pre-blooming, fruit set, veraison) have been conducted. Results indicated a good efficacy of pyraclostrobin, alone or in tank mixture with copper, and of the mixture trifloxystrobin+tebuconazole in reducing the disease. Moreover, the same fungicides significantly reduced the inoculum density of Colletotrichum spp. on fruit surface and the incidence of latent infections, as compared to the traditional copper applications.

"Mal secco", an Italian name meaning "dry disease", is a severe tracheomycotic disease of citrus caused by the mitosporic fungus Phoma tracheiphila (Petri) Kantsch. et Gik. It appeared in 1894 in two Aegean Greek islands, from which it spread almost to the whole Mediterranean basin and the Black Sea. Due to its high susceptibility, lemon is the most damaged citrus species. Disease damage consists of substantial reduction of the quality and quantity of the crop, mainly due to the difficulties of controlling the disease and the replacement of susceptible valuable cultivars by others which are less vulnerable, but have low productivity and scarce fruit quality. Control of mal secco disease has relied on a number of diverse chemical and nonchemical strategies, but is still faced with efficacy problems. Host resistance remains a most desirable goal, but it will not be ultimately achieved until the genetic basis of resistance to P tracheiphila are not fully elucidated. The present paper reviews the different aspects of citrus mal secco as studied worldwide over almost a century of research, from the first appeareance of the disease in Italy (1918) to date. Milestones and pitfalls about the symptomatology, aetiology, host-parasite relationship, diagnosis, epidemiology, and control are discussed in a historical perspective, emphasizing the advancements in knowledge. Finally, some issues and challenges are highlighted that need to be more comprehensively addressed prior to deployment of effective disease control measures.

A survey on Phytophthora spp. in the soils and roots of citrus groves was carried out in the main Syrian growing areas of Lattakia and Tartous. Traditional assays (selective medium with soil dilution plates) were used for pathogen detection, and molecular (PCR) assays were used for unanmbiguous identification of P. nicotianae and P. citrophthora in 38.5% of the collected samples. In both locations, P. citrophthora was the predominant species.

Capnodis tenebrionis (L.) (Coleoptera: Buprestidae), the so called Mediterranean flat-headed root-borer, is an economically important phytophagous pest species mainly on stone fruit trees (apricot, plum, cherry, peach and nectarine). Chemicals and Entomopathogenic nematodes are used for the control of adults and neonate larvae, respectively. Further control means are under investigations in order to have more options within Integrated Pest Control strategies. This study is aimed at investigating the susceptibility of rootstocks to the larvae of C. tenebrionis. Two bioassays were carried out during 2016 and 2017. A first bioassay was based on the evaluation of a potential antixenosis action expressed by neonate larvae infesting twigs of rootstocks (Marianna 26, Barrier, Adesoto, Mylaboran 29C, GF677, Garnem, Cab 6P, Max Ma60 and Colt). This bioassay allowed to process a high number of different rootstocks in a short time. It has a preliminary value. The second bioassay assessed the antibiosis influence of the rootstocks through the breeding of larvae (since the neonate ones) on artificial diets containing bark flour of Adesoto, Cab 6P, Colt, Garnem, GF677, Max Ma60, Montclar and 29C rootstock. The first bioassay showed that Colt, Mylaboran 29C and GF677 were the most susceptible rootstocks to larval infestation of C. tenebrionis and Max Ma60 was less favorable to the pest. Concerning the effects of the diet, larvae reared on a diet containing Montclar, Cab 6P and GF 677 bark flour had a mean daily increase of their weight higher that those reared on cortex tissues of other genotypes whereas Garnem and Colt had a lower increase.

Two organic amendments (OMW-M1 and 11 OMW-M2), based on olive mill wastes (OMWs) subjected to advanced processes of aerobic static storage or composting, were tested for their suppressive activity against Verticillium dahliae, the causal agent of olive Verticillium wilt. OMW-M1 and OMW-M2 drastically inhibited the pathogen growth in vitro and then were further tested in suppressive pot experiments. The amendments, mixed at 15 % (v/v) with a nursery standard plant-growth matrix, were tested alone or in combination with two biocontrol bacteria (Bacillus amyloliquefaciens and Burkholderia cepacia) selected from suppressive soils. All mixtures were artificially contaminated with V. dahliae microsclerotia (MS), the density of which was periodically monitored by either a semi-selective medium or a specific real-time PCR technique. In plant-less pot assays conducted in a growth chamber, OMW-M1 was the most effective amendment, reducing V. dahliae MS density by 100 %after 90 days with respect to the untreated control. In nursery experiments with pot-growing olive plants, OMW-M1, particularly when combined with the biocontrol bacteria, confirmed its strong suppressive activity reducing up to 100 % the density of V. dahliae MS in the rhizosphere behaving even better than a commercial biofungicide (Trichoderma asperellum TV1)used as a control. The best combined treatment also reduced plant mortality and increased root and shoot extension. We conclude that organic amendments from stabilized olive mill by-products showed positive agronomic and phyto-sanitary properties on pot-growing olive plants and, particularly when enriched with biocontrol agents, they are potentially suitable for use in sustainable agriculture.

The selection of appropriate plant species is critical in the successful application of phytoremediation techniques. The present study is an attempt to assess the capability of three brassicaceae, Brassica alba (L.) Rabenh, Brassica carinata A. Braun and Brassica nigra (L.) Koch, for the phytoextraction of Cr, Cu, Pb and Zn from an unpolluted and polluted silty loamy soil added with either Bacillus licheniformis BLMB1 or compost or both. Experiments were conducted in a greenhouse in pots filled with the soils. In all experiments metals were shown to accumulate in shoots and roots of plants grown on polluted soils, and both compost and B. licheniformis BLMB1 strain were able to enhance the accumulation of metals, especially Cr. In particular, Cr accumulation in B. alba resulted higher than the Cr threshold for hyperaccumulator plants (1000 mg kg−1). This result provides a new plant resource that may have a potential use for phytoextraction of Cr from contaminated soil. However, because of the low bioconcentration factors (b1) for all studied metals, these species cannot be regarded as suitable for the phytoextraction of excessive Cr, Cu, Pb and Zn from polluted soils. Thus, these species may be used with success only for low metal polluted soils.

Xylella fastidiosa has been reported as responsible for a devastating disease on olive trees in Apulia region (south-eastern Italy), characterized by a quick decline syndrome. In Lebanon, the pathogen was recently associated with leaf scorch symptoms on oleander, and reports on leaf scorch and dieback of olive trees branches by technicians and farmers have shown an increasing trend in the main agricultural areas. To assess the occurrence and distribution of the pathogen in Lebanon, samples of twigs from olive trees (82), olive seedlings (26), grapevine (30), oleander (32) and ornamentals imported from Italy (48) were analysed by isolation on four agarized media, serological techniques (ELISA and DTBIA) using Xylella fastidiosa -specific antibodies and by PCR, using three specific sets of primers. Results unequivocally demonstrated that all the collected samples were free from the pathogen. As well, both detection protocols and attempts at isolating the pathogen on agarized media demonstrated that oleander samples gathered from American University campus in Beirut, where X. fastidiosa was previously reported, were not infected. Nevertheless, continuous monitoring and rigorous control measures of propagative materials are necessary to prevent the introduction of Xylella fastidiosa in Lebanon.