Effects of Biochar on the Growth and Development of Tomato Seedlings and on the Response of Tomato Plants to the Infection of Systemic Viral Agents.

Biochar is a rich carbon product obtained by pyrolysis of biomass under a limited supply of oxygen. It is composed mainly of aromatic molecules, but its agronomic value is hard to evaluate and difficult to predict due to its great variable characteristics depending on the type of starting biomass and the conditions of pyrolysis. Anyway, it could be used as soil amendment because it increases the soil fertility of acidic soils, increases the agricultural productivity, and seems to provide protection against some foliar and soilborne diseases. In this study, the effects of biochar, obtained from olive pruning, have been evaluated on tomato seedlings growth and on their response to systemic agents' infection alone or added with beneficial microorganisms (Bacillus spp. and Trichoderma spp.). First, experimental data showed that biochar seems to promote the development of the tomato seedlings, especially at concentrations ranging from 1 to 20% (w/w with peat) without showing any antimicrobial effects on the beneficial soil bacteria at the tomato rhizosphere level and even improving their growth. Thus, those concentrations were used in growing tomato plants experimentally infected with tomato spotted wilt virus (TSWV) and potato spindle tuber viroid (PSTVd). The biochar effect was estimated by evaluating three parameters, namely, symptom expression, number of infected plants, and pathogen quantification, using RT-qPCR technique and -ΔΔCt analysis. Biochar at 10-15% and when added with Trichoderma spp. showed that it reduces the replication of PSTVd and the expression of symptoms even if it was not able to block the start of infection. The results obtained on TSWV-infected plants suggested that biochar could contribute to reducing both infection rate and virus replication. For systemic viral agents, such as PSTVd and TSWV, there are no curative control methods, and therefore, the use of prevention means, as can be assumed the use biochar, for example, in the nursery specialized in horticultural crops, can be of great help. These results can be an encouraging starting point to introduce complex biochar formulates among the sustainable managing strategies of plant systemic diseases.

A non-targeted metabolomics study on Xylella fastidiosa infected olive plants grown under controlled conditions.

In the last decade, the bacterial pathogen Xylella fastidiosa has devastated olive trees throughout Apulia region (Southern Italy) in the form of the disease called "Olive Quick Decline Syndrome" (OQDS). This study describes changes in the metabolic profile due to the infection by X. fastidiosa subsp. pauca ST53 in artificially inoculated young olive plants of the susceptible variety Cellina di Nardò. The test plants, grown in a thermo-conditioned greenhouse, were also co-inoculated with some xylem-inhabiting fungi known to largely occur in OQDS-affected trees, in order to partially reproduce field conditions in terms of biotic stress. The investigations were performed by combining NMR spectroscopy and MS spectrometry with a non-targeted approach for the analysis of leaf extracts. Statistical analysis revealed that Xylella-infected plants were characterized by higher amounts of malic acid, formic acid, mannitol, and sucrose than in Xylella-non-infected ones, whereas it revealed slightly lower amounts of oleuropein. Attention was paid to mannitol which may play a central role in sustaining the survival of the olive tree against bacterial infection. This study contributes to describe a set of metabolites playing a possible role as markers in the infections by X. fastidiosa in olive.

An hydroxytyrosol enriched extract from olive mill wastewaters exerts antioxidant activity and antimicrobial activity on Pseudomonas savastanoi pv. savastanoi and Agrobacterium tumefaciens.

An hydroxytyrosol (HTyr) enriched extract (HTE), obtained from olive mill wastewaters (OMWW) through an eco-friendly, patented process was tested on two olive tree pathogens, Pseudomonas savastanoi pv. savastanoi (Pss) and Agrobacterium tumefaciens (At). Pss, in particular, is a feared treat for olive tree cultivation being the etiological agent of the olive knot disease responsible of severe production losses. Chemical characterization allowed to identify and quantify HTyr as the main constituent along with other low molecular weight phenols. HTE has proven potent antioxidant activity and significant antimicrobial activity against Pss and At in vitro, in both cases higher than hydroxytyrosol alone, suggesting an important role also of the minor phenolic components, which act synergistically with HTyr. Alternatives to the molecules currently present on the market are needed, but only the use of standardized extraction technologies would allow to conduct solid field studies and enable this circular approach to find a real application in the olive groves.

Effect of potassium sorbate (E-202) and the antifungal PgAFP protein on Aspergillus carbonarius growth and ochratoxin A production in raisin simulating media.

BACKGROUND: Ochratoxin A (OTA) is a mycotoxin produced by several species of Aspergillus and Penicillium fungi. The presence of OTA in raisins is mainly related to black Aspergillus spp. contamination. This toxin poses risks to human and animal health due to its high toxicity and carcinogenicity. New strategies to avoid the risk associated with OTA are therefore necessary. RESULTS: A comparison was made between the effects of the antifungal protein PgAFP and potassium sorbate (E-202) on the growth of Aspergillus carbonarius, biosynthetic- and stress-related gene expression and its OTA production at two water activity (aw ) levels, 0.95 and 0.93 aw . The results showed that PgAFP successfully controlled OTA production, whereas E-202, despite being able to reduce Aspergillus carbonarius growth, caused a significant increase in OTA production by the fungus. CONCLUSION: PgAFP protein, a biological compound with an antifungal activity, is safer to use than E-202 and may be proposed as a food preservative and a useful strategy to control ochratoxigenic A. carbonarius in raisins. © 2018 Society of Chemical Industry.

Development of Real-Time Isothermal Amplification Assays for On-Site Detection of Phytophthora infestans in Potato Leaves.

Real-time loop-mediated isothermal amplification (LAMP) and recombinase polymerase amplification (RPA) assays were developed targeting the internal transcribed spacer 2 region of the ribosomal DNA of Phytophthora infestans, the potato late blight causal agent. A rapid crude plant extract (CPE) preparation method from infected potato leaves was developed for on-site testing. The assay's specificity was tested using several species of Phytophthora and other potato fungal and oomycete pathogens. Both LAMP and RPA assays showed specificity to P. infestans but also to the closely related species P. andina, P. mirabilis, P. phaseoli, and P. ipomoeae, although the latter are not reported as potato pathogen species. No cross-reaction occurred with P. capsici or with the potato pathogens tested, including P. nicotianae and P. erythroseptica. The sensitivity was determined using P. infestans pure genomic DNA added into healthy CPE samples. Both LAMP and RPA assays detected DNA at 50 fg/µl and were insensitive to CPE inhibition. The isothermal assays were tested with artificially inoculated and naturally infected potato plants using a Smart-DART platform. The LAMP assay effectively detected P. infestans in symptomless potato leaves as soon as 24 h postinoculation. A rapid and accurate on-site detection of P. infestans in plant material using the LAMP assay will contribute to improved late blight diagnosis and early detection of infections and facilitate prompt management decisions.

Seasonal Fluctuations of Sap-Feeding Insect Species Infected by Xylella fastidiosa in Apulian Olive Groves of Southern Italy.

A study on seasonal abundance of Auchenorrhyncha species and their infectivity by Xylella fastidiosa in the Apulia region of Italy was conducted to identify ideal periods for monitoring and adoption of potential control measures against insect vectors. Adult populations of Auchenorrhyncha species were monitored monthly over a 2-yr period from five olive groves. A total of 15 species were captured, identified, and tested for presence of X. fastidiosa by polymerase chain reaction (PCR). For three species, Philaenus spumarius L., Neophilaenus campestris (Fallèn), and Euscelis lineolatus Brullé, positive reactions to X. fastidiosa were obtained, on average, in 16.3, 15.9 and 18.4% of adult insects, respectively. Philaneous spumarius was the dominant species (39.8% of total Auchenorrhyncha captured) with the highest adult abundance in summer months. Adult P. spumarius and N. campestris were first detected between March and May in both years, and all insects tested during these periods (year 1: n = 42, year 2: n = 132) gave negative reactions to X. fastidiosa by PCR. Similarly, first adults of E. lineolatus that appeared from October to November (year 1: n = 20, year 2: n = 15) tested negative for presence of X. fastidiosa Given the lack of transstadial and transovarial transmission of X. fastidiosa and considering that P. spumarius is univoltine, control measures against nymphal stages of P. spumarius should be investigated as means of population suppression to reduce spread of X. fastidiosa in olive groves.

Extensive Field Survey, Laboratory and Greenhouse Studies Reveal Complex Nature of Pseudomonas syringae-Associated Hazelnut Decline in Central Italy.

Pseudomonas avellanae (Pav) has been reported as the causal agent of bacterial decline and bacterial canker of hazelnut in Italy and Greece, respectively. Both hazelnut diseases were reported to be similar in terms of symptoms, severity and persistence. In this study, we found that both symptomatic and asymptomatic trees in the field were colonized by Pav. Multilocus Sequence Typing (MLST) analysis showed that Pav strains isolated during this study in Italy belong to the P. syringae phylogroup 1 and they are closely related to Pav strains previously isolated in Greece from hazelnut bacterial canker. On the other hand, strains isolated in earlier studies from hazelnut decline in Italy belong to both phylogroup 1 and 2 of P. syringae. Both phylogroup 1 strains of P. syringae from Greece and Italy are different than strains isolated in this study in terms of their capacity to excrete fluorescent pigments on different media. Despite the same plant genotype and cropping practices adopted, the incidence of hazelnut decline ranged from nearly 0 to 91% across our study sites. No disease developed on plants inoculated with Pav through wounding while leaf scar inoculations produced only mild disease symptoms. Based on our results and the previously reported correlation between pedo-climatic conditions and hazelnut decline, we conclude that hazelnut decline in central Italy could be incited by a combination of predisposing (adverse pedo-climatic conditions) and contributing factors (Pav). Because this is a true decline different from "bacterial canker" described in Greece, we refer to it as hazelnut decline (HD).

Whole-Genome Sequencing of 10 Pseudomonas syringae Strains Representing Different Host Range Spectra.

Pseudomonas syringae is a ubiquitous bacterium that readily persists in environmental habitats as a saprophyte and also is responsible for numerous diseases of crops. Here, we report the whole-genome sequences of 10 strains isolated from both woody and herbaceous plants that will contribute to the elucidation of the determinants of their host ranges.

Mutability in Pseudomonas viridiflava as a programmed balance between antibiotic resistance and pathogenicity.

Mutable bacterial cells are defective in their DNA repair system and often have a phenotype different from that of their wild-type counterparts. In human bacterial pathogens, the mutable and hypermutable phenotypes are often associated with general antibiotic resistance. Here, we quantified the occurrence of mutable cells in Pseudomonas viridiflava, a phytopathogenic bacterium in the P. syringae complex with a broad host range and capacity to live as a saprophyte. Two phenotypic variants (transparent and mucoid) were produced by this bacterium. The transparent variant had a mutator phenotype, showed general antibiotic resistance and could not induce disease on the plant species tested (bean). In contrast, the mucoid variant did not display mutability or resistance to antibiotics and was capable of inducing disease on bean. Both the transparent and mucoid variants were less fit when grown in vitro, whereas, in planta, both of the variants and wild-types attained similar population densities. Given the importance of the methyl-directed mismatch repair system (MMR) in the occurrence of mutable and hypermutable cells in human bacterial pathogens, we investigated whether mutations in mut genes were associated with mutator transparent cells in P. viridiflava. Our results showed no mutations in MMR genes in any of the P. viridiflava cells tested. Here, we report that a high mutation rate and antibiotic resistance are inversely correlated with pathogenicity in P. viridiflava, but are not associated with mutations in MMR. In addition, P. viridiflava variants differ from variants produced by other phytopathogenic bacteria in the absence of reversion to the wild-type phenotype.

A framework to gauge the epidemic potential of plant pathogens in environmental reservoirs: the example of kiwifruit canker.

New economically important diseases on crops and forest trees emerge recurrently. An understanding of where new pathogenic lines come from and how they evolve is fundamental for the deployment of accurate surveillance methods. We used kiwifruit bacterial canker as a model to assess the importance of potential reservoirs of new pathogenic lineages. The current kiwifruit canker epidemic is at least the fourth outbreak of the disease on kiwifruit caused by Pseudomonas syringae in the mere 50 years in which this crop has been cultivated worldwide, with each outbreak being caused by different genetic lines of the bacterium. Here, we ask whether strains in natural (non-agricultural) environments could cause future epidemics of canker on kiwifruit. To answer this question, we evaluated the pathogenicity, endophytic colonization capacity and competitiveness on kiwifruit of P. syringae strains genetically similar to epidemic strains and originally isolated from aquatic and subalpine habitats. All environmental strains possessing an operon involved in the degradation of aromatic compounds via the catechol pathway grew endophytically and caused symptoms in kiwifruit vascular tissue. Environmental and epidemic strains showed a wide host range, revealing their potential as future pathogens of a variety of hosts. Environmental strains co-existed endophytically with CFBP 7286, an epidemic strain, and shared about 20 virulence genes, but were missing six virulence genes found in all epidemic strains. By identifying the specific gene content in genetic backgrounds similar to known epidemic strains, we developed criteria to assess the epidemic potential and to survey for such strains as a means of forecasting and managing disease emergence.

The Pseudomonas viridiflava phylogroups in the P. syringae species complex are characterized by genetic variability and phenotypic plasticity of pathogenicity-related traits.

As a species complex, Pseudomonas syringae exists in both agriculture and natural aquatic habitats. P.viridiflava, a member of this complex, has been reported to be phenotypically largely homogenous. We characterized strains from different habitats, selected based on their genetic similarity to previously described P.viridiflava strains. We revealed two distinct phylogroups and two different kinds of variability in phenotypic traits and genomic content. The strains exhibited phase variation in phenotypes including pathogenicity and soft rot on potato. We showed that the presence of two configurations of the Type III Secretion System [single (S-PAI) and tripartite (T-PAI) pathogenicity islands] are not correlated with pathogenicity or with the capacity to induce soft rot in contrast to previous reports. The presence/absence of the avrE effector gene was the only trait we found to be correlated with pathogenicity of P.viridiflava. Other Type III secretion effector genes were not correlated with pathogenicity. A genomic region resembling an exchangeable effector locus (EEL) was found in S-PAI strains, and a probable recombination between the two PAIs is described. The ensemble of the variability observed in these phylogroups of P.syringae likely contributes to their adaptability to alternating opportunities for pathogenicity or saprophytic survival.

Summer heat and low soil organic matter influence severity of hazelnut Cytospora canker.

Cytospora canker, caused by the fungus Cytospora corylicola, is present in hazelnut production areas worldwide. The disease is widespread throughout the main production areas of Italy. The causal agent is considered to be a secondary invader of damaged tissue that attacks mainly stressed plants. However, little is known of disease severity and stress factors that predispose plants to infection. In particular, the role of pedoclimatic factors was investigated. Direct survey indicated that disease severity varied across several study sites. Geostatistics showed a strong positive correlation between disease severity index and summer heat (r = 0.80 and 0.91 for July and August, respectively) and strong negative correlation between disease severity index and soil organic matter (r = -0.78). A moderate positive correlation between disease severity index and magnesium/potassium ratio (r = 0.58) and moderate negative correlations between disease severity index and total soil nitrogen (r = -0.53), thermal shock (r = -0.46), and rainfall (r = -0.53) were determined. No significant correlation between disease severity index and soil aluminum (r = -0.35), soil pH (r = -0.01), and plant age (r = -0.38) was found.

Epidemiological study of hazelnut bacterial blight in central Italy by using laboratory analysis and geostatistics.

Incidence of Xanthomonas arboricola pv. corylina, the causal agent of hazelnut bacterial blight, was analyzed spatially in relation to the pedoclimatic factors. Hazelnut grown in twelve municipalities situated in the province of Viterbo, central Italy was studied. A consistent number of bacterial isolates were obtained from the infected tissues of hazelnut collected in three years (2010-2012). The isolates, characterized by phenotypic tests, did not show any difference among them. Spatial patterns of pedoclimatic data, analyzed by geostatistics showed a strong positive correlation of disease incidence with higher values of rainfall, thermal shock and soil nitrogen; a weak positive correlation with soil aluminium content and a strong negative correlation with the values of Mg/K ratio. No correlation of the disease incidence was found with soil pH. Disease incidence ranged from very low (<1%) to very high (almost 75%) across the orchards. Young plants (4-year old) were the most affected by the disease confirming a weak negative correlation of the disease incidence with plant age. Plant cultivars did not show any difference in susceptibility to the pathogen. Possible role of climate change on the epidemiology of the disease is discussed. Improved management practices are recommended for effective control of the disease.