Deep Learning Based Barley Disease Quantification for Sustainable Crop Production.

Net blotch disease caused by Drechslera teres is a major fungal disease that affects barley (Hordeum vulgare) plants and can result in significant crop losses. In this study, we developed a deep-learning model to quantify net blotch disease symptoms on different days post-infection on seedling leaves using Cascade R-CNN (Region-Based Convolutional Neural Networks) and U-Net (a convolutional neural network) architectures. We used a dataset of barley leaf images with annotations of net blotch disease to train and evaluate the model. The model achieved an accuracy of 95% for cascade R-CNN in net blotch disease detection and a Jaccard index score of 0.99, indicating high accuracy in disease quantification and location. The combination of Cascade R-CNN and U-Net architectures improved the detection of small and irregularly shaped lesions in the images at 4-days post infection, leading to better disease quantification. To validate the model developed, we compared the results obtained by automated measurement with a classical method (necrosis diameter measurement) and a pathogen detection by real-time PCR. The proposed deep learning model could be used in automated systems for disease quantification and to screen the efficacy of potential biocontrol agents to protect against disease.

Long-read PacBio genome sequencing of four environmental saprophytic Sporothrix species spanning the pathogenic clade.

BACKGROUND: The genus Sporothrix belongs to the order Ophiostomatales and contains mainly saprobic soil and plant fungi, although pathogenic species capable of causing human infections are also present. The whole-genomes of disease-causing species have already been sequenced and annotated but no comprehensive genomic resources for environmental Sporothrix species are available, thus limiting our understanding of the evolutionary origin of virulence-related genes and pathogenicity. RESULT: The genome assembly of four environmental Sporothrix species resulted in genome size of ~ 30.9 Mbp in Sporothrix phasma, ~ 35 Mbp in S. curviconia, ~ 38.7 Mbp in S. protearum, and ~ 39 Mbp in S. variecibatus, with a variable gene content, ranging from 8142 (S. phasma) to 9502 (S. variecibatus). The analysis of mobile genetic elements showed significant differences in the content of transposable elements within the sequenced genomes, with the genome of S. phasma lacking several class I and class II transposons, compared to the other Sporothrix genomes investigated. Moreover, the comparative analysis of orthologous genes shared by clinical and environmental Sporothrix genomes revealed the presence of 3622 orthogroups shared by all species, whereas over 4200 genes were species-specific single-copy gene products. Carbohydrate-active enzyme analysis revealed a total of 2608 protein-coding genes containing single and/or multiple CAZy domains, resulting in no statistically significant differences among pathogenic and environmental species. Nevertheless, some families were not found in clinical species. Furthermore, for each sequenced Sporothrix species, the mitochondrial genomes was assembled in a single circular DNA molecule, ranging from 25,765 bp (S. variecibatus) to 58,395 bp (S. phasma). CONCLUSION: In this study, we present four annotated genome assemblies generated using PacBio SMRT sequencing data from four environmental species: S. curviconia, S. phasma, S. protearum and S. variecibatus with the aim to provide a starting point for future comparative genome evolution studies addressing species diversification, ecological/host adaptation and origin of pathogenic lineages within the genus Sporothrix.

Screening and molecular characterization of new thermo- and ethanol-tolerant Acetobacter malorum strains isolated from two biomes Moroccan cactus fruits.

Industrially, the sensitivity of acetic acid bacteria (AAB) to the high temperatures and the high ethanol concentrations is the major concerns for manufacturers. This study was conceived and designed to isolate and identify new thermo- and ethanol-tolerant AAB from Opuntia ficus-indica L. fruits. As a result, among 140 isolated bacterial strains, five selected strains (CR1, CR5, CR23, CZ2, and CZ15) exhibited important acetic acid production until 40 °C. The use of 16S rDNA gene analysis was insufficient to identify selected bacteria. Indeed, except CR5 that presented 100% similarity to A. cerevisiae, the other strains presented similar homology rates simultaneously to the 16S rDNA sequences of A. cerevisiae and A. malorum. The reidentification by 16S-23S rDNA gene sequencing showed that CR1, CR23, and CZ15 were A. malorum, which were shown tolerance to the highest concentration of ethanol (12%) and produced elevated amount (40 g/L) of acetic acid at 37 °C. In summary, we showed the thermotolerance and ethanol tolerant character of new A. malorum strains, which can be used as a starter for vinegar production. Furthermore, during the molecular characterization of the isolated strains, we concluded that 16S-23S rDNA internal transcribed spacer sequence is of great importance for discriminating between AAB species as a complement to the identification by 16S rDNA sequencing.

Differentiated dynamics of bud dormancy and growth in temperate fruit trees relating to bud phenology adaptation, the case of apple and almond trees.

Few studies have focused on the characterization of bud dormancy and growth dynamics for temperate fruit species in temperate and mild cropping areas, although this is an appropriate framework to anticipate phenology adaptation facing future warming contexts which would potentially combine chill declines and heat increases. To examine this issue, two experimental approaches and field observations were used for high- and low-chill apple cultivars in temperate climate of southern France and in mild climates of northern Morocco and southern Brazil. Low-chill almond cultivars offered an additional relevant plant material for comparison with apple in northern Morocco. Divergent patterns of dormancy and growth dynamics were clearly found in apple tree between southern France and southern Brazil. Divergences were less pronounced between France and Morocco. A global view outlined main differences in the dormancy chronology and intensity, the transition between endordormancy and ecodormancy and the duration of ecodormancy. A key role of bud rehydration in the transition period was shown. High-chill cultivars would be submitted in mild conditions to heterogeneous rehydration capacities linked to insufficient chill fulfillment and excessive forcing linked to high temperatures. This would favor bud competitions and consequently excessive flowering durations and weak flowering. Low chilling requirements in apple and almond would conversely confer biological capacities to tolerate superficial dormancy and abrupt transition from endordormancy to ecodormancy without important heterogeneous rehydration states within buds. It may also assume that low-chill cultivars can also tolerate high temperatures during ecodormancy as well as extended flowering durations.

Exploring Active Peptides with Antimicrobial Activity In Planta against Xylella fastidiosa.

Xylella fastidiosa (Xf) is a xylem-limited quarantine plant bacterium and one of the most harmful agricultural pathogens across the world. Despite significant research efforts, neither a direct treatment nor an efficient strategy has yet been developed for combatting Xylella-associated diseases. Antimicrobial peptides (AMPs) have been gaining interest as a promising sustainable tool to control pathogens due to their unique mechanism of action, broad spectrum of activity, and low environmental impact. In this study, we disclose the bioactivity of nine AMPs reported in the literature to be efficient against human and plant pathogen bacteria, i.e., Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa, against Xf, through in vitro and in vivo experiments. Based on viable-quantitative PCR (v-qPCR), fluorescence microscopy (FM), optical density (OD), and transmission electron microscopy (TEM) assays, peptides Ascaphin-8 (GF19), DASamP1 (FF13), and DASamP2 (IL14) demonstrated the highest bactericidal and antibiofilm activities and were more efficient than the peptide PB178 (KL29), reported as one of the most potent AMPs against Xf at present. Furthermore, these AMPs showed low to no toxicity when tested on eukaryotic cells. In in planta tests, no Xf disease symptoms were noticed in Nicotiana tabacum plants treated with the AMPs 40 days post inoculation. This study highlighted the high antagonistic activity of newly tested AMP candidates against Xf, which could lead to the development of promising eco-friendly management of Xf-related diseases.

Screening and Characterization of New Acetobacter fabarum and Acetobacter pasteurianus Strains with High Ethanol−Thermo Tolerance and the Optimization of Acetic Acid Production

The production of vinegar on an industrial scale from different raw materials is subject to constraints, notably the low tolerance of acetic acid bacteria (AAB) to high temperatures and high ethanol concentrations. In this study, we used 25 samples of different fruits from seven Moroccan biotopes with arid and semi-arid environmental conditions as a basic substrate to isolate thermo- and ethanol-tolerant AAB strains. The isolation and morphological, biochemical and metabolic characterization of these bacteria allowed us to isolate a total number of 400 strains with characters similar to AAB, of which six strains (FAGD1, FAGD10, FAGD18 and GCM2, GCM4, GCM15) were found to be mobile and immobile Gram-negative bacteria with ellipsoidal rod-shaped colonies that clustered in pairs and in isolated chains. These strains are capable of producing acetic acid from ethanol, growing on peptone and oxidizing acetate to CO2 and H2O. Strains FAGD1, FAGD10 and FAGD18 show negative growth on YPG medium containing D-glucose > 30%, while strains GCM2, GCM4 and GCM15 show positive growth. These six strains stand out on CARR indicator medium as isolates of the genus Acetobacter ssp. Analysis of 16S rDNA gene sequencing allowed us to differentiate these strains as Acetobacter fabarum and Acetobacter pasteurianus. The study of the tolerance of these six isolates towards pH showed that most of the six strains are unable to grow at pH 3 and pH 9, with an ideal pH of 5. The behavior of the six strains at different concentrations of ethanol shows an optimal production of acetic acid after incubation at concentrations between 6% and 8% (v/v) of ethanol. All six strains tolerated an ethanol concentration of 16% (v/v). The resistance of the strains to acetic acid differs between the species of AAB. The optimum acetic acid production is obtained at a concentration of 1% (v/v) for the strains of FAGD1, FAGD10 and FAGD18, and 3% (v/v) for GCM2, GCM4 and GCM15. These strains are able to tolerate an acetic acid concentration of up to 6% (v/v). The production kinetics of the six strains show the highest levels of growth and acetic acid production at 30 °C. This rate of growth and acetic acid production is high at 35 °C, 37 °C and 40 °C. Above 40 °C, the production of acid is reduced. All six strains continue to produce acetic acid, even at high temperatures up to 48 °C. These strains can be used in the vinegar production industry to minimize the load on cooling systems, especially in countries with high summer temperatures.

Beneficial Microorganisms to Control the Gray Mold of Grapevine: From Screening to Mechanisms.

In many vineyards around the world, Botrytis cinerea (B. cinerea) causes one of the most serious diseases of aerial grapevine (Vitis vinifera L.) organs. The control of the disease relies mainly on the use of chemical products whose use is increasingly challenged. To develop new sustainable methods to better resist B. cinerea, beneficial bacteria were isolated from vineyard soil. Once screened based on their antimicrobial effect through an in vivo test, two bacterial strains, S3 and S6, were able to restrict the development of the pathogen and significantly reduced the Botrytis-related necrosis. The photosynthesis analysis showed that the antagonistic strains also prevent grapevines from considerable irreversible PSII photo-inhibition four days after infection with B. cinerea. The 16S rRNA gene sequences of S3 exhibited 100% similarity to Bacillus velezensis, whereas S6 had 98.5% similarity to Enterobacter cloacae. On the other hand, the in silico analysis of the whole genome of isolated strains has revealed the presence of "biocontrol-related" genes supporting their plant growth and biocontrol activities. The study concludes that those bacteria could be potentially useful as a suitable biocontrol agent in harvested grapevine.

Looking for New Antifungal Drugs from Flavonoids: Impact of the Genetic Diversity of Candida albicans on the in-vitro Response.

BACKGROUND: In an era in which antimicrobial resistance is increasing at an alarming pace, it is very important to find new antimicrobial agents effective against pathogenic microrganisms resistant to traditional treatments. Among the notable breakthroughs in the past years of research in natural-drug discovery, there is the identification and testing of flavonoids, a group of plant-derived substances capable of promoting many beneficial effects on humans. These compounds show different biological activities such as inhibition of neuroinflammation and tumor growth as well as antimicrobial activity against many microbial pathogens. METHODS: We undertook a review of protocols and standard strains used in studies reporting the inhibitory effects of flavonoids against Candida albicans by focusing our attention on genetic characterization of the strains examined. Moreover, using the C. albicans MLST-database, we performed a phylogenetic analysis showing the genetic variation occurring in this species. RESULTS: Today, we have enough information to estimate genetic diversity within microbial species and recent data revealed that most of fungal pathogens show complex population structures in which not a single isolate can be designated as representative of the entire taxon. This is especially true for the highly divergent fungal pathogen C. albicans, in which the assumption that one or few "standard strains" can represent the whole species is overly unrealistic and should be laid to rest. CONCLUSION: The goal of this article is to shed light on the extent of genetic variation in C. albicans and how this phenomenon can largely influence the activity of flavonoids against this species.

Adaptation of Phytophthora infestans to Partial Resistance in Potato: Evidence from French and Moroccan Populations.

ABSTRACT The use of partially resistant cultivars should become an essential component of a sustainable management strategy of potato late blight, caused by Phytophthora infestans. It is therefore important to determine to what extent P. infestans populations can be selected for increased aggressiveness by potato cultivars with different levels of partial resistance. To this end, we sampled P. infestans populations from France and Morocco, chosen as locations where late blight occurs regularly but which differ in the distribution of potato cultivars. Cross-inoculation experiments were used to determine the aggressiveness of all populations to potato cvs. Bintje (prevalent in France but not grown in Morocco) and Désirée (popular in Morocco but cultivated to a very small extent in France). French populations were more aggressive on cv. Bintje than on cv. Désirée, irrespective of the site they were sampled from. Their aggressiveness increased between early and late samplings, suggesting that both cultivars selected for increased aggressiveness during epidemics. By contrast, Moroccan populations were more aggressive on Désirée, regarded as partially resistant in Europe, than on Bintje, highly susceptible under European conditions. These data indicate that P. infestans populations adapt to locally dominant cultivars, irrespective of their resistance levels, and can therefore overcome polygenic, partial resistance. This adaptive pattern may render partial resistance nondurable if not properly managed.

Molecular characterization of patulin producing and non-producing Penicillium species in apples from Morocco.

The isolation of patulin-producing Penicillia in apples collected in different markets in four localities in Morocco is reported. Fungi were identified by ß-tubulin sequencing and further characterized using a specific PCR-based method targeting the isoepoxydon dehydrogenase (IDH) gene to discriminate between patulin-producing and non-producing strains. Production of patulin was also evaluated using standard cultural and biochemical methods. Results showed that 79.5% of contaminant fungi belonged to the genus Penicillium and that Penicillium expansum was the most isolated species (83.9%) followed by Penicillium chrysogenum (~9.7%) and Penicillium crustosum (~6.4%). Molecular analysis revealed that 64.5% of the Penicillium species produced the expected IDH-amplicon denoting patulin production in these strains. However, patulin production was not chemically confirmed in all P. expansum strains. The isolation of IDH(-)/patulin(+) strains poses the hypothesis that gentisylaldehyde is not a direct patulin precursor, supporting previous observations that highlighted the importance of the gentisyl alcohol in the production of this mycotoxin. Total agreement between IDH-gene detection and cultural/chemical methods employed was observed in 58% of P. expansum strains and for 100% of the other species isolated. Overall the data reported here showed a substantial genetic variability within P. expansum population from Morocco.

Arthrographis curvata and Rhodosporidium babjevae as New Potential Fungal Lipase Producers for Biotechnological Applications

Abstract Fungi have always attracted a lot of attention as they are able to produce a vast repertoire of enzymes that find a broad spectrum of uses in biotechnological and industrial fields. Undoubtedly, one of the most promising biocatalysts is the lipase, which has been widely used for the biotransformation of a number of commercial products due to its high stability, high catalytic efficiency, versatility and selectivity, making it one of the most attractive and best-studied enzymes. In this study we report the isolation and molecular identification of new lipase-producing fungi from different environmental samples from Morocco. The production and activity of extracellular lipases, at different parameters, was evaluated using the Rhodamine B agar, submerged fermentation and biochemical methods. Two fungal strains Arthrographis curvata and Rhodosporidium babjevae, were isolated and found to produce large amounts of lipases. The optimal activity of the extracellular lipase was detected at 40°C and pH 9.0 for A. curvata and at 40 °C and pH 8.0 for R. babjevae. This study add new information at the growing list of fungal species producing lipases with improved physicochemical proprieties which could constitute a new line of research for further studies and to be exploited for industrial or bioremediation purposes.