Influence of chitin nanofibers and gallic acid on physical-chemical and biological performances of chitosan-based films.

In this work, chitosan films loaded with gallic acid and different content of chitin nanofibers were prepared and subjected to different characterization techniques. The results showed that the inclusion of gallic acid to chitosan films caused moderate decrease in water vapor permeability (by 29 %) and increased tensile strength of films (by 169 %) in comparison to the neat chitosan films. Furthermore, it was found that the addition of chitin nanofibers up to 30 % into chitosan/gallic acid films additionally improved tensile strength (by 474 %) and reduced plasticity of films (by 171 %), when compared to the chitosan/gallic acid films. Increased concentration of chitin nanofibers in films reduced the overall water vapor permeability of films by 51 %. In addition, gallic acid and chitin nanofibers had synergic effect on high chitosan film's antioxidant and antifungal activity toward Botrytis cinerea (both above 95 %). Finally, chitosan/gallic acid/chitin nanofibers films reduced decay incidence of strawberries, increased total soluble solid content, and promoted high production of some polyphenols during cold storage, in comparison to the control chitosan films and uncoated strawberry samples. Hence, these results suggest that chitosan/gallic acid/chitin nanofibers can present eco-sustainable approach for preservation of strawberries, giving them additional nutritional value.

Use of Pseudomonas protegens to Control Root Rot Disease Caused by Boeremia exigua var. exigua in Industrial Chicory (Cichorium intybus var. sativum Bisch.).

Boeremia exigua var. exigua is a recurrent pathogen causing root rot in industrial chicory. Currently, there is no chemical or varietal control for this disease, and thus, management strategies need to be developed. This study determined the biocontrol effect of strains of Pseudomonas protegens bacteria with antimicrobial compounds on the fungus B. exigua var. exigua under in vitro, in vivo, and field conditions. In addition, root colonization by these bacteria was estimated by the phlD-specific PCR-based dilution end point assay. Eighteen isolates of Pseudomonas spp were evaluated, and the strains that showed the greatest in vitro inhibition of fungal mycelial growth (mm), Ca10A and ChB7, were selected. Inoculation with the strain ChB7 showed less severity (necrotic area) under in vivo conditions (root trials) compared with the control inoculated with the pathogen (p ≤ 0.05). The molecular analysis revealed that the root colonization of plants grown in pots was equal to or greater than 70%. Similar levels were observed in the field trials conducted at the Selva Negra and Canteras experimental stations (2015-2016 season), with values ranging from 85.7 to 70.5% and from 75.0 to 79.5%, respectively. Regarding yield (ton ha-1), values were higher in the treatments inoculated with strains Ca10A and ChB7 (p ≤ 0.05) at both experimental sites, while a lower incidence and severity of root rot were observed at Selva Negra. These results suggest that the Chilean strains of P. protegens are a promising tool for the control of root diseases in industrial chicory.

Integrative Pathogenicity Assay and Operational Taxonomy-Based Detection of New Forma Specialis of Fusarium oxysporum Causing Datepalm Wilt.

Pathogenicity-associated genes are highly host-specific and contribute to host-specific virulence. We tailored the traditional Koch's postulates with integrative omics by hypothesizing that the effector genes associated with host-pathogenicity are determinant markers for virulence, and developed Integrative Pathogenicity (IP) postulates for authenticated pathogenicity testing in plants. To set the criteria, we experimented on datepalm (Phoenix dactylifera) for the vascular wilt pathogen and confirmed the pathogen based on secreted in xylem genes (effectors genes) using genomic and transcriptomic approaches, and found it a reliable solution when pathogenicity is in question. The genic regions ITS, TEF1-α, and RPBII of Fusarium isolates were examined by phylogenetic analysis to unveil the validated operational taxonomy at the species level. The hierarchical tree generated through phylogenetic analysis declared the fungal pathogen as Fusarium oxysporum. Moreover, the Fusarium isolates were investigated at the subspecies level by probing the IGS, TEF1-α, and Pgx4 genic regions to detect the forma specialis of F. oxysporum that causes wilt in datepalm. The phylogram revealed a new forma specialis in F. oxysporum that causes vascular wilt in datepalm.

Trichoderma spp. Genes Involved in the Biocontrol Activity Against Rhizoctonia solani.

Rhizoctonia solani is a pathogen that causes considerable harm to plants worldwide. In the absence of hosts, R. solani survives in the soil by forming sclerotia, and management methods, such as cultivar breeding, crop rotations, and fungicide sprays, are insufficient and/or inefficient in controlling R. solani. One of the most challenging problems facing agriculture in the twenty-first century besides with the impact of global warming. Environmentally friendly techniques of crop production and improved agricultural practices are essential for long-term food security. Trichoderma spp. could serve as an excellent example of a model fungus to enhance crop productivity in a sustainable way. Among biocontrol mechanisms, mycoparasitism, competition, and antibiosis are the fundamental mechanisms by which Trichoderma spp. defend against R. solani, thereby preventing or obstructing its proliferation. Additionally, Trichoderma spp. induce a mixed induced systemic resistance (ISR) or systemic acquired resistance (SAR) in plants against R. solani, known as Trichoderma-ISR. Stimulation of every biocontrol mechanism involves Trichoderma spp. genes responsible for encoding secondary metabolites, siderophores, signaling molecules, enzymes for cell wall degradation, and plant growth regulators. Rhizoctonia solani biological control through genes of Trichoderma spp. is summarized in this paper. It also gives information on the Trichoderma-ISR in plants against R. solani. Nonetheless, fast-paced current research on Trichoderma spp. is required to properly utilize their true potential against diseases caused by R. solani.

First report of Diplodia mutila causing wood necrosis in European hazelnut in Chile.

In Chile, the planted area of European hazelnut (Corylus avellana L.) reaches around 30,000 hectares, mainly concentrated in the central and southern area of the country where climate and soil provide a natural environment well suited to growing this species. Only a few diseases affect this nut tree in Chile. During the spring seasons in 2018 and 2020, European hazelnut plants (6 to 20% of incidence) exhibited wood necrosis and vascular discoloration of branches, with reduced growth, cankers and wilt branches, in orchards located in San Clemente and Curicó, Maule Region, Bulnes and El Carmen, Ñuble Region, Chile (36°45'-36°54' S; 71°03'-72°26' W). Symptomatic tissues were surface disinfected using a ~1% commercial sodium hypochlorite solution. Disinfected tissues were cut longitudinally, placed onto potato dextrose agar (PDA, Difco) plates, and incubated at 25 °C in the dark for 48 hours. Fungal hyphal tips were taken and placed on PDA medium. A fungal species was consistently isolated from these lignified tissues. The mycelium was initially translucent (turning white in appearance), while the mature mycelium was aerial, varying in color from pale to dark gray (Munsell color code: colony edge mycelium 6Y-6 4 / 5G and colony center mycelium B6-PB 7 / 5PB). The production of pycnidia and conidia was induced using pine needles in water agar medium and incubated in the dark for 10 days. Hyaline unicellular conidia of 25 ± 1.1 µm (range 23.9 to 26.1 µm) long and 11 ± 0.5 µm (Range 10.5 to 11.5 µm) wide (n = 50) were obtained from black pycnidia. Based on the cultural and morphological characteristics observed, the pathogen was identified as a possible species of the family Botryosphaeriaceae (20 isolates). Molecular techniques were used to identify the species of pathogen, and three isolates (F154, F199, and F167) were analyzed by using Multilocus sequence typing to confirm the identity of the pathogen. Genes ITS (internal transcribed spacer region), tef-1 (translation elongation factor 1-alpha) and ß-tub (ß-tubulin) were amplified using endpoint PCR, with primers ITS1/ITS4 (White et al., 1990), EF1-728F/EF1-986R (Carbone & Kohn, 1999) and Bt2a/Bt2b (Glass & Donaldson, 1995), respectively. The segments were sequenced using the same primers, deposited in Gen Bank, and the accession numbers for each isolate were OM993582, OM993583, ON003481 for ITS, ON054936, ON054938, ON054937 for tef1 and ON054939, ON054941, ON054940 for ß-tub, respectively. A phylogenetic tree was constructed using the maximum likelihood statistical method with the Tamura-Nei model based on a concatenated dataset of ITS region, tef1 and ß-tubulin gene using Mega-X, and the three Chilean isolates (F154, F199, and F167) formed a single clade with the reference isolates of Diplodia mutila (Fr.) Mont. BLAST algorithm analyses indicated 100% identity to D. mutila for ITS (accession NR_144906), for tef-1 (accession MK573559), and for ß-tubulin (accession MG952719). The pathogenicity of the three isolates was validated through Koch's postulates. For this purpose, a trial was established in 6-year-old European hazelnut plants cv. Tonda Di Giffoni. Ten healthy branches were individually inoculated using actively growing mycelial discs from each isolate, while a disc of PDA without fungus was used as a control. Holes of 5-mm diameter were inoculated, making sure the mycelium was in contact with the wood. Finally, the wounds were sealed with plastic film to prevent external contamination and improve humidity conditions. After 120 days, each branch was cut longitudinal-sectioned to verify the presence of wood necrosis which arose between 3.0 to 16.2 mm of length around the point of inoculation. No necrosis was observed in the control. To confirm pathogenicity, infected tissues were cut into small pieces with sterile knives and scalpels, and surface disinfected with a 1% sodium hypochlorite solution for 1 min. The disinfected tissues were placed on PDA medium and incubated at 25°C in the dark until fungal growth was observed. Hyphal tips were taken from the mycelia developed from the pieces of wood, and placed on PDA medium in order to obtain pure isolates. The pathogenicity of the D. mutila isolates F154 and F199 was observed in 100% of the inoculated branches, while isolate F167 showed symptoms in 85% of the branches. The reisolated strains showed similar mycelial growth and microscopic fungal structures to those observed in the isolates used for inoculation. This is the first report of D. mutila affecting European hazelnut in Chile. This fungus has been recently reported affecting hazelnut in Oregon, USA (Wiman et al., 2019), causing similar symptoms to those observed in our study. In addition, D. mutila has been reported infecting walnut in Chile (Diaz et al. 2018) and native forest trees, specifically Araucaria araucana in Chile (Besoain et al., 2017). The presence of D. mutila in commercial hazelnut orchards in Chile highlights the need for epidemiological studies in order to understand the characteristics and impact of this pathogen and, based on this, develop adequate phytosanitary programs for its control.

Antagonistic Activity of Chilean Strains of Pseudomonas protegens Against Fungi Causing Crown and Root Rot of Wheat (Triticum aestivum L.).

Seed treatments with antagonistic bacteria could reduce the severity of crown and root rot diseases in wheat crops. The objective of this study was to evaluate the potential antagonistic activity of a bacterial consortium of three Chilean strains of Pseudomonas protegens against the wheat crown and root rot pathogens Gaeumannomyces graminis var. tritici, Rhizoctonia cerealis, and Fusarium culmorum. Two field experiments were carried out on artificially infested soil during two consecutive seasons (2016-2017 and 2017-2018) in an Andisol soil of southern Chile. Control treatments (not inoculated with fungi) were also included. Each treatment included a seed treatment of spring wheat cv. Pantera-INIA with and without the bacterial consortium. Both phytosanitary damage (incidence and severity) and agronomic components were evaluated. Bacterial populations with the phlD+ gene in the wheat plant rhizosphere during anthesis state (Z6) were also quantified. In both seasons, infection severity decreased by an average of 16.8% in seeds treated with P. protegens consortium, while yield components such as spikes m-1 and number of grains per spike increased. The use of antagonistic bacteria resulted in a total yield increase only during the first experimental season (P < 0.05). In general, accumulated rainfall influenced the antagonistic effect of the consortium of P. protegens strains, accounting for the differences observed between the two seasons. The results suggest that this P. protegens consortium applied on seeds can promote plant growth and protect wheat crops against crown and root rot pathogens in Southern Chile under field conditions.

Distribution and Prevalence of Fusarium Crown Rot and Common Root Rot Pathogens of Wheat in Montana.

Distribution of Fusarium crown rot (FCR) and common root rot (CRR) pathogens associated with wheat (Triticum aestivum) in 91 fields in Montana were determined during the 2008 and 2009 crop seasons using real-time quantitative polymerase chain reaction (qPCR) and conventional isolation methods. Correlations (P < 0.001) were found between detection methods for both diseases. FCR was detected in 57% of the fields and CRR was detected in 93% of the fields surveyed. Percent incidence based on isolation from individual tillers was Bipolaris sorokiniana (15%), F. culmorum (13%), and F. pseudograminearum (8%). FCR populations were highly variable across the regions and were not detected in any fields from the Gb5 soil types of Judith Basin and Fergus counties. The spatial distributions of FCR and CRR were affected by elevation, soil type, and temperature. High FCR populations were associated with spring wheat crops rather than winter wheat based on qPCR (P < 0.001). FCR and CRR could produce yield losses in a range of 3 to 35%. This study is the first time that qPCR was used to survey these two pathogen groups, and the merits and weakness of qPCR relative to traditional isolation methods are discussed.

Population Dynamics Between Fusarium pseudograminearum and Bipolaris sorokiniana in Wheat Stems Using Real-Time qPCR.

Fusarium pseudograminearum and Bipolaris sorokiniana are causal agents of Fusarium crown rot and common root rot, respectively, of wheat and cause significant losses worldwide. Understanding the population dynamics between these two pathogens at late stages of wheat development is needed. The effect of F. pseudograminearum and B. sorokiniana inocula applied singly or in mixtures at seeding to spring wheat 'Hank' was measured using seedling stand, grain yield, and pathogen populations in the first internode at heading, milk, and harvest stage of wheat development using real-time quantitative polymerase chain reaction. High and low rates of F. pseudograminearum inoculum reduced B. sorokiniana populations in field trials but B. sorokiniana inoculations did not affect F. pseudograminearum populations. Populations of both pathogens increased from heading until harvest, with F. pseudograminearum colonizing lower internodes earlier than B. sorokiniana. Neither pathogen prevented infection by the other in the first internode of wheat stems. Inoculations increased incidence of infection and co-infection relative to natural settings observed for both pathogens. At the seedling stage, both fungi, individually or combined, reduced the seedling stands when compared with a noninoculated control for the three location-years. Grain yield and F. pseudograminearum populations were inversely correlated, while B. sorokiniana populations were not correlated with yield.