Pomegranate Rhizosphere Microbial Diversity Revealed by Metagenomics: Toward Organic Farming, Plant Growth Promotion and Biocontrol?

Food production must undergo systems change to meet the sustainable development goals (SDGs). For example, organic farming can be empowered by soil microorganisms with plant growth promotion (PGP) and biocontrol features. In this context, there have been limited studies on pomegranate. We investigated microbial diversity in rhizosphere of the pomegranate "Bhagwa" variety and its potential role in PGP and biocontrol. Both bulk and rhizosphere soil samples were analyzed for their physicochemical properties. Whole metagenome sequencing was conducted using the Illumina NovaSeq6000 platform. Surprisingly, we found that bulk and rhizosphere soil samples had comparable microbial diversity. Metagenome sequencing revealed the abundance of Streptomyces indicus, Bradyrhizobium kalamazoonesis, and Pseudomonas cellulosum in the rhizosphere that are reported here for the first time in agricultural literature. Pathway prediction analysis using KEGG (Kyoto Encyclopedia for Genes and Genomes) and COG (clusters of orthologous genes) databases identified metabolic pathways associated with biocontrol properties against pathogens. We confirmed the metagenome data in vitro, which demonstrated their PGP potential and antimicrobial properties. For instance, S. indicus produced high concentration of indole-3-acetic acid, a PGP phytohormone, that can stimulate plant growth. In addition, an antimicrobial susceptibility assay suggested that bacterial extracts displayed activity against Xanthomonas, a primary pathogen causing the pomegranate wilt disease. In conclusion, this study suggests that S. indicus, B. kalamazoonesis, and P. cellulosum can potentially be PGP and biocontrol agents that may contribute to increased crop productivity in pomegranate cultivation. These agents and their combinations warrant future research with an eye on SDGs and so as to enable and innovate organic farming and pomegranate agricultural practices.

Optimization of Fermentation Process of Pomegranate Peel and Schisandra Chinensis and the Biological Activities of Fermentation Broth: Antioxidant Activity and Protective Effect Against H2O2-induced Oxidative Damage in HaCaT Cells.

In this study, the lactobacillus fermentation process of pomegranate (Punica granatum L.) peel and Schisandra chinensis (Turcz.) Baill (PP&SC) was optimized by using the response surface method (RSM) coupled with a Box-Behnken design. The optimum fermentation condition with the maximal yield of ellagic acid (99.49 ± 0.47 mg/g) was as follows: 1:1 (w:w) ratio of pomegranate peel to Schisandra chinensis, 1% (v:v) of strains with a 1:1 (v:v) ratio of Lactobacillus Plantarum to Streptococcus Thermophilus, a 37 °C fermentation temperature, 33 h of fermentation time, 1:20 (g:mL) of a solid-liquid ratio and 3 g/100 mL of a glucose dosage. Under these conditions, the achieved fermentation broth (FB) showed stronger free radical scavenging abilities than the water extract (WE) against the ABTS+, DPPH, OH- and O2- radicals. The cytotoxicity and the protective effect of FB on the intracellular ROS level in HaCaT cells were further detected by the Cell Counting Kit-8 (CCK-8) assay. The results showed that FB had no significant cytotoxicity toward HaCaT cells when its content was no more than 8 mg/mL. The FB with a concentration of 8 mg/mL had a good protective effect against oxidative damage, which can effectively reduce the ROS level to 125.94% ± 13.46% (p < 0.001) compared with 294.49% ± 11.54% of the control group in H2O2-damaged HaCaT cells. The outstanding antioxidant ability and protective effect against H2O2-induced oxidative damage in HaCaT cells promote the potential for the FB of PP&SC as a functional raw material of cosmetics.

Diversity and biopotential of Bacillus velezensis strains A6 and P42 against rice blast and bacterial blight of pomegranate.

Bacillus velezensis is widely known for its inherent biosynthetic potential to produce a wide range of bio-macromolecules and secondary metabolites, including polyketides (PKs) and siderophores, as well as ribosomally and non-ribosomally synthesized peptides. In the present study, we aimed to investigate the bio-macromolecules, such as proteins and peptides of Bacillus velezensis strains, namely A6 and P42 by whole-cell sequencing and highlighted the potential application in controlling phytopathogens. The bioactive compounds, specifically secondary metabolites, were characterized by whole-cell protein profiling, Thin-Layer Chromatography, Infra-Red Spectroscopy, Nuclear Magnetic Resonance, Gas Chromatograph and Electro Spray Liquid Chromatography. Gas Chromatography analysis revealed that the A6 and P42 strains exert different functional groups of compounds, such as aromatic ring, aliphatic, alkene, ketone, amine groups and carboxylic acid. Whole-cell protein profiling of A6 and P42 strains of B. velezensis by nano-ESI LC-MS/MS revealed the presence of 945 and 5303 proteins, respectively. The in vitro evaluation of crude extracts (10%) of A6 and P42 significantly inhibited the rice pathogen, Magnaporthe oryzae (MG01), whereas the cell-free culture filtrate (75%) of strain P42 showed 58.97% inhibition. Similarly, in vitro evaluation of crude extract (10%) of P42 strain inhibited bacterial blight of pomegranate pathogen, Xanthomonas axonopodis pv. punicae, which eventually resulted in a higher inhibition zone of 3 cm, whereas the cell-free extract (75%) of the same strain significantly suppressed the growth of the pathogen with an inhibition zone of 1.48 cm. From the results obtained, the crude secondary metabolites and cell-free filtrates (containing bio-macromolecules) of the strains A6 and P42 of B. velezensis can be employed for controlling the bacterial and fungal pathogens of crop plants.

Agrobacterium tumefaciens-mediated transformation of Coniella granati.

Pomegranate fruit rot caused by Coniella granati is among the most devastating diseases threatening pomegranate production. The pathogenic mechanism of this pathogen remains largely unknown due to lack of genetic transformation method. Herein, we developed an approach to the Agrobacterium tumefaciens-mediated transformation (ATMT) of C. granati using a plasmid vector encoding the green fluorescent protein (GFP) and hygromycin resistance (Hyg) genes. This approach yielded C. granati transformants that exhibited uniform, stable green fluorescence. We further optimized this ATMT protocol, enabling us to achieve a transformation efficiency of up to 300 transformants per 0.5 cm2 mycelial plug. Together, we thus provide the first report of the stable transformation of C. granati, laying a foundation for future functional studies characterizing this economically important fungal pathogen.

Ultrasonic potential in maintaining the quality and reducing the microbial load of minimally processed pomegranate.

Difficulty of Pomegranate fruit peeling and arils separation are the main motivations of progressive ready-to-eat pomegranate fresh arils industry. Also, extracted pomegranate arils are highly perishable due to water loss and microbial contamination expose. The aim of the current work was then to evaluate the effect of ultrasound for 15 and 30 min on maintenance of bioactive compounds and microbial load reduction of pomegranate arils cv. Rabbab. Treated arils were kept at 5 °C and analyzed during 15 days of storage. The most and least weight loss of arils obtained in control and 30 min treated samples, respectively. After 12 days of storage, all samples were decayed except those treated for 15 and 30 min. The ultrasound treatment significantly prevented degradation of anthocyanin and ascorbic acid compounds. Total phenol and antioxidant activity decreased during storage. At the end of storage, the most total phenol content (3898.6 mg GAE L-1) was found in arils treated for 30 min whereas the most anthocyanin (91.93 mg L-1), total antioxidant activity (82.65%), and ascorbic acid (2.53 mg L-1) were found in arils treated for 15 min. Ultrasound treated arils had lower microbial load (total mesophilic bacteria in control and 30-min treated samples) in each stage during storage. At the end of storage, the microbial load in treated and control arils was 0.7 and 0.2 Log CFU g-1, respectively). Overall, ultrasound treatment effectively reduced weight loss and preserved bioactive compounds during storage.

Polyhydroxyalkanoate synthesis by bacteria isolated from landfill and ETP with pomegranate peels as carbon source.

Many bacteria produce polyhydroxyalkanoates (PHAs) when exposed to stressful conditions. It is a known fact that PHAs have the potential to replace petrochemical-based plastics as they are biodegradable, biocompatible, and thermoprocessible materials. The study focusses on producing PHA from microbes isolated from polluted environments and pomegranate peels were utilized as a unique carbon source. This was done to ensure reduction in the cost of the substrate that has not yet been reported as a substrate for PHA production. A comparative study was also done with Cupriavidus necator, the reference strain. Out of many bacterial strains, isolated, eight of these were found to have ability to produce PHA. Pomegranate peel was substituted as carbon source in the medium and all bacterial isolates along with reference strain were used to test their ability to produce PHA from waste. Cupriavidus necator, the reference strain, yielded 71% PHA. Bacillus halotolerans DSM8802 yielded 83% at 1:1:: C:N ratio at 72 h.

[Protection by some plant methanol extracts of cherry tomatoes (Solanum lycopersicum var. Cerasiforme) from fungic infection by Alternaria alternata]. / Utilisation d'extraits méthanoliques de plantes pour la protection des cultures de tomates-cerises (Solanum lycopersicum var. cerasiforme) contre l'infection fongique par Alternaria alternata.

Cherry tomato is very susceptible to fungal infections that can cause considerable damage in crops and during storage. Alternaria infection is one of the most common and dangerous alterations for this fruit. They are caused by Alternaria alternata or some other species belonging to the same genus. In this work, we tested the antifungal activity of methanol extracts from five plants harvested in the region of Jijel (Algeria) on A. alternata. The activity was first tested in vitro and then on greenhouse cherry tomato plants: extracts were applied to healthy plants before infection in order to test their preventive action, and after infection to determine whether they are able to knock out Alternaria. Results showed that Rosmarinus officinalis and Lavandula angustifolia extracts were the most active in vitro on A. alternata. Microscopic observations of the mold indicated that these extracts inhibited the dictyospores production. The antifungal activity tested on the plants grown in greenhouse revealed that R. officinalis extract still was the most active. Extracts of L. angustifolia and Punica granatum did not protect the plants from Alternaria infection, but provided a total cure at the end of the treatment. Extracts from Quercus suber and Eucalyptus globulus were the least active. They did not bestow any protection nor complete healing of the plants. Dictyospores counting on fruits at the end of the treatment confirmed the results obtained for the greenhouse crops.

TITLE: Utilisation d'extraits méthanoliques de plantes pour la protection des cultures de tomates-cerises (Solanum lycopersicum var. cerasiforme) contre l'infection fongique par Alternaria alternata. ABSTRACT: La tomate-cerise est un fruit très sujet aux infections fongiques qui peuvent causer des dégâts considérables dans les cultures et lors de la conservation. Les alternarioses comptent parmi les altérations les plus répandues et dangereuses pour ce fruit. Elles sont causées par Alternaria alternata ou d'autres espèces appartenant au même genre. Dans ce travail, nous avons testé l'activité antifongique d'extraits méthanoliques de cinq plantes récoltées dans la région de Jijel (Algérie) sur A. alternata. L'activité a d'abord été testée in vitro, puis sur des plants de tomates-cerises cultivés sous serre : les extraits ont été appliqués sur des plants sains, avant l'infection, afin de tester leur action préventive, et après l'infection pour déterminer s'ils sont capables de traiter l'alternariose. Les résultats ont montré que les extraits de Rosmarinus officinalis et Lavandula angustifolia étaient les plus actifs in vitro sur A. alternata. L'observation microscopique de la moisissure a indiqué que ces extraits agissaient en inhibant sa production de dictyospores. L'activité antifongique testée sur les plants cultivés sous serre a révélé que l'extrait de R. officinalis était toujours le plus actif. Venaient ensuite les extraits de L. angustifolia et Punica granatum qui n'ont pas permis la protection des plants contre l'alternariose, mais qui ont néanmoins donné une guérison totale à la fin du traitement. Les extraits de Quercus suber et Eucalyptus globulus étaient les moins actifs. Ils n'ont permis ni la prévention, ni la guérison complète des plants. Le comptage des dictyospores réalisé sur les fruits à la fin du traitement a confirmé les résultats obtenus pour les cultures sous serre.

Xanthomonas axonopodis pv. punicae depends on multiple non-TAL (Xop) T3SS effectors for its coveted growth inside the pomegranate plant through repressing the immune responses during bacterial blight development.

Xanthomonas axonopodis pv. punicae (Xap), the bacterial blight pathogen of pomegranate, incurs substantial loss to yield and reduces export quality of this economically important fruit crop. During infection, the bacterium secretes six non-TAL (Xop) effectors into the pomegranate cells through a specialized type three secretion system (T3SS). Previously, we demonstrated the role of two key effectors, XopL and XopN in pathogenesis. Here, we investigate the role of rest effectors (XopC2, XopE1, XopQ and XopZ) on disease development. We generated null mutants for each individual effector and mutant bacterial suspension was infiltrated into pomegranate leaves. Compared to Xap wild, the mutant bacterial growth was reduced by 2.7-11.5 folds. The mutants produced lesser water-soaked lesions when infiltrated on leaves by 1.13-2.21 folds. Among the four effectors, XopC2 contributes highest for in planta bacterial growth and disease development. XopC2 efficiently suppressed the defense responses like callose deposition, reactive oxygen species (ROS) and the activation of immune responsive genes. Being a major contributor, we further characterize XopC2 for its subcellular localization, its protein structure and networking. XopC2 is localized to the plasma membrane of Nicotiana benthamiana like XopL and XopN. XopC2 is a 661 amino acids protein having 15 alpha and 17 beta helix. Our STRING and I-TASSER based analysis hinted that XopC2 interacts with multiple membrane localized plant proteins including transcription regulator of CCR4-NOT family, TTN of maintenance of chromosome family and serine/threonine-protein phosphatase 2A (PP2A) isoform. Based on the interaction it is predicted that XopC2 might involve in diverse functions like nuclear-transcribed mRNA catabolic process, maintenance of chromosome, hormone signaling and protein dephosphorylation activities and thereby suppress the plant immunity. Altogether, our study suggests that Xap largely depends on three non-TAL (Xop) effectors, including XopC2, XopL and XopN, to modulate pomegranate PTI for its unrestricted proliferation during bacterial blight development.

Microbial, nutritional, and organoleptic quality of pomegranate juice following high-pressure homogenization and low-temperature pasteurization.

This study investigated the effects of high-pressure homogenization (HPH) on microbial, nutritional, and organoleptic qualities of pomegranate juice. Juices subjected to HPH at 100 and 150 MPa were compared to those subjected to thermal pasteurization at 55, 65, and 75 °C for 15 s, and to combined treatments. Physicochemical properties of pomegranate juices, such as color, pH, acidity, and total soluble solids, were marginally affected by either treatment. Significant microbial inactivation for the juices inoculated with Escherichia coli and yeast was achieved during 28 days of shelf life by HPH at 150 MPa, followed by thermal treatment at 65 °C. There was no significant difference in ascorbic acid levels between the treated and fresh juice. Total polyphenol content and antioxidant activity were measured to be higher in case of both treatment methods. Analyzing treated HPH samples with electronic tongue and nose showed similar flavor profiles to the fresh juice. PRACTICAL APPLICATION: The work shows the benefits of using the advanced high-pressure homogenization (HPH) compared to common thermal treatment used today in fruit juice pasteurization. HPH is capable to provide sufficient reduction in microbial with better nutritional and organoleptic quality. In this manuscript, we showed the potential of HPH as alternative method for pasteurization on a highly healthy fruit like pomegranate. The work describes the advantages in HPH process through comprehensive analysis for nutritional, microbial, and sensorial quality.

The Effect of Polyphenols on Pomegranate Fruit Susceptibility to Pilidiella granati Provides Insights into Disease Tolerance Mechanisms.

Pilidiella granati, also known as Coniella granati, is the etiological agent of pomegranate fruit dry rot. This fungal pathogen is also well-known as responsible for both plant collar rot and leaf spot. Because of its aggressiveness and the worldwide diffusion of pomegranate crops, the selection of cultivars less susceptible to this pathogen might represent an interesting preventive control measure. In the present investigation, the role of polyphenols in the susceptibility to P. granati of the two royalties-free pomegranate cultivars Wonderful and Mollar de Elche was compared. Pomegranate fruit were artificially inoculated and lesion diameters were monitored. Furthermore, pathogen DNA was quantified at 12-72 h post-inoculation within fruit rind by a real time PCR assay setup herein, and host total RNA was used in expression assays of genes involved in host-pathogen interaction. Similarly, protein extracts were employed to assess the specific activity of enzymes implicated in defense mechanisms. Pomegranate phenolic compounds were evaluated by HPLC-ESI-MS and MS2. All these data highlighted 'Wonderful' as less susceptible to P. granati than 'Mollar de Elche'. In the first cultivar, the fungal growth seemed controlled by the activation of the phenylpropanoid pathway, the production of ROS, and the alteration of fungal cell wall. Furthermore, antifungal compounds seemed to accumulate in 'Wonderful' fruit following inoculation. These data suggest that pomegranate polyphenols have a protective effect against P. granati infection and their content might represent a relevant parameter in the selection of the most suitable cultivars to reduce the economic losses caused by this pathogen.

Characterization of Penicillium s.s. and Aspergillus sect. nigri causing postharvest rots of pomegranate fruit in Southern Italy.

Most of diseases of pomegranate fruit are caused by fungal pathogens, which provoke postharvest yield and economical losses. Aspergillus and Penicillium sensu lato (s.l.) are the main wound pathogens of pomegranate fruit. In the present investigation, the populations of Aspergillus and Penicillium s.l. isolated from pomegranate fruit in Southern Italy were characterized. Since the morphological identification of species belonging to these genera is laborious, molecular approaches, such as PCR and High-Resolution Melting (HRM), were used. Particularly, a specific primer pair was designed to discriminate, within the Penicillium s.l. population, Penicillium sensu stricto (s.s.) from Talaromyces strains. Then, a new HRM assay for species identification within Penicillium s.s. according to SNPs present in a portion of the beta-tubulin gene was set up. Similarly, Aspergillus sect. nigri population was characterized arranging a HRM assay, whose primer pair was designed on a portion of the calmodulin gene. According to these assays, 10% of the Penicillium s.l. population proved to be made up of Talaromyces biverticillius strains. Furthermore, six species of Penicillium s.s. (P. adametzioides, P. brevicompactum, P. citrinum, P. glabrum, P. pagulum, and P. johnkrugii) and four of black aspergilli (A. tubingensis, A. welwitschiae, A. japonicus, and A. uvarum) were identified; all species belonging to both genera disclosed different incidences in postharvest rotted pomegranate fruit. Moreover, since Aspergillus and Penicillium are potentially producers of mycotoxins, like ochratoxin A and fumonisins, the presence/absence of genes involved in mycotoxin biosynthetic pathways was tested. Some Aspergillus strains belonging to species A. welwitschiae proved to possess fumonisin genes. The setup of molecular tools to characterize Penicillium s.l. and Aspergillus sect. nigri species infecting pomegranate fruit after harvest is of paramount importance for their effective control, even more considering the ability of these fungal genera to produce mycotoxins, which are hazardous for human health and potentially present also in by-products.

Effect of temperature and pH on the probiotication of Punica granatum juice using Lactobacillus species.

This study was focused on the effects of fermentation temperature and pH on the quality of Punica granatum juice probioticated with Lactobacillus species: Lactobacillus plantarum, Lactobacillus casei, Lactobacillus bulgaricus, and Lactobacillus salivarius. The whole fruit juice of P. granatum which is rich with phytonutrients appeared to be a good probiotic carrier. The probiotication was carried out for 24 hr at 30, 35, and 37°C and pH 2.5, 4.0, and 5.5 under microaerophilic conditions. The results found that P. granatum juice cultivated with L. casei had a better growth profile with a higher biomass density at 37°C around pH 3.5-4.0. Probiotication could maintain the scavenging activity of P. granatum juice cultivated with L. casei. The scavenging activity achieved up to 90% inhibition at the concentration of 5 mg/ml. The whole fruit-squeezed P. granatum juice was suitable for the growth of Lactobacillus species even without supplementation during cultivation. PRACTICAL APPLICATIONS: The findings of this study presented the potential of P. granatum juice (whole fruit) to be used as a good probiotic carrier, particularly for Lactobacillus species without supplementation. High nutritious P. granatum juice catered the need of probiotic bacteria during fermentation. Probiotication could maintain the antioxidant capacity of the juice in term of its radical scavenging activity. The antioxidant capacity was mainly attributed to the metabolites such as phenolic acids (romarinic acid and caftaric acid) and flavonoids (quercetin, quercetin 3-glucoside, rutin and kaempferol rutinoside). With the optimized temperature (37°C) and pH (4.00), probiotic bacteria could growth well up to a cell viability of 2.46 × 1010  cfu/ml. This offers P. granatum to be developed into functional food to cater to the needs of the consumers who are lactose intolerant to dairy products.

Reliable and early diagnosis of bacterial blight in pomegranate caused by Xanthomonas axonopodis pv. punicae using sensitive PCR techniques.

Bacterial blight caused by Xanthomonas axonopodis pv. punicae is a major disease of pomegranate. Bacterial blight drastically reduces the yield and quality of fruits, which are critical for pomegranate production. Precise and early diagnosis of bacterial blight is crucial for active surveillance and effective management of the disease. Symptoms based disease diagnostic methods are labor-intensive, time-consuming and may not detect disease on asymptomatic plants. DNA-based disease diagnostics using polymerase chain reaction (PCR) are reliable, precise, accurate and quick. PCR coupled with agarose gel electrophoresis (PCR-AGE), PCR coupled with capillary electrophoresis (PCR-CE) and real-time PCR (qPCR) were applied for the early and accurate diagnosis of bacterial blight in pomegranate. PCR-CE and qPCR were capable of diagnosing bacterial blight 6 to 10 days before symptom appearance, with detection limits of 100 fg and 10 fg of bacterial DNA respectively. However, conventional PCR-AGE detected pathogen at the onset of disease symptoms with a detection limit of 10 pg of bacterial DNA. qPCR detected bacterial blight in orchards that did not show any disease symptoms. Our data demonstrate that qPCR is more sensitive than other PCR methods along with being reliable for early diagnosis.