Functional Hyperspectral Imaging by High-Related Vegetation Indices to Track the Wide-Spectrum Trichoderma Biocontrol Activity Against Soil-Borne Diseases of Baby-Leaf Vegetables

Research has been increasingly focusing on the selection of novel and effective biological control agents (BCAs) against soil-borne plant pathogens. The large-scale application of BCAs requires fast and robust screening methods for the evaluation of the efficacy of high numbers of candidates. In this context, the digital technologies can be applied not only for early disease detection but also for rapid performance analyses of BCAs. The present study investigates the ability of different Trichoderma spp. to contain the development of main baby-leaf vegetable pathogens and applies functional plant imaging to select the best performing antagonists against multiple pathosystems. Specifically, sixteen different Trichoderma spp. strains were characterized both in vivo and in vitro for their ability to contain R. solani, S. sclerotiorum and S. rolfsii development. All Trichoderma spp. showed, in vitro significant radial growth inhibition of the target phytopathogens. Furthermore, biocontrol trials were performed on wild rocket, green and red baby lettuces infected, respectively, with R. solani, S. sclerotiorum and S. rolfsii. The plant status was monitored by using hyperspectral imaging. Two strains, Tl35 and Ta56, belonging to T. longibrachiatum and T. atroviride species, significantly reduced disease incidence and severity (DI and DSI) in the three pathosystems. Vegetation indices, calculated on the hyperspectral data extracted from the images of plant-Trichoderma-pathogen interaction, proved to be suitable to refer about the plant health status. Four of them (OSAVI, SAVI, TSAVI and TVI) were found informative for all the pathosystems analyzed, resulting closely correlated to DSI according to significant changes in the spectral signatures among health, infected and bio-protected plants. Findings clearly indicate the possibility to promote sustainable disease management of crops by applying digital plant imaging as large-scale screening method of BCAs' effectiveness and precision biological control support

Managing Rhizoctonia Damping-Off of Rocket (Eruca sativa) Seedlings by Drench Application of Bioactive Potato Leaf Phytochemical Extracts

Plants produce a huge array of secondary metabolites that play a key role in defense mechanisms against detrimental microorganisms and herbivores, and represent a suitable alternative to synthetic fungicides in sustainable agriculture. In this work, twelve crude hydroethanolic extracts derived from leaves of different potato cultivars were chemically characterized by LC/MS and their antioxidant properties were investigated in vitro. Furthermore, the biological activity against the fungal pathogen Rhizoctonia solani was evaluated both in vitro and in vivo. Extracts showed the ability to inhibit R. solani growth in vitro and significantly reduced damping-off incidence in in vivo experiments. Furthermore, R. solani mycelia exposed to the extracts showed an altered morphology (low translucency, irregular silhouette, and cytoplasmatic content coagulation) compared to the untreated control in light microscopy examination. Principal component analysis conducted on identified chemical compounds highlighted significant metabolic variations across the different extracts. In particular, those that inhibited most of the growth of the pathogen were found to be enriched in α-chaconine or α-solanine content, indicating that their biological activity is affected by the abundance of these metabolites. These results clearly indicated that plant-derived compounds represent a suitable alternative to chemicals and could lead to the development of new formulates for sustainable control of plant diseases

ACTIVITY OF CHESTNUT TANNINS AGAINST THE SOUTHERNROOT-KNOT NEMATODE MELOIDOGYNE INCOGNITA

Studies on the effects of tannins on plant-parasitic nematodes are few. A new formulation of a hydrolysable tanninextracted from chestnut (SaviotaN®) was tested for efficacy in controlling Meloidogyne incognita. Therefore, in vitro andpot experiments on tomato were performed to investigate the nematicidal activity of tannin aqueous solutions at differentconcentrations on M. incognita. In the in vitro experiment the following concentrations of tannin at 0.30, 0.40, 0.50, 0.75,1.00, 1.25, 1.50 g L−1 were tested for their effect on the nematode. The second-stage juveniles (J2s) immobility increasedwith increasing concentration and exposure time. All tested tannin concentrations were effective to reduce viability fromabout 45 to 70% after 10 days of exposure, in comparison to the treated and untreated controls. The immobile J2s recoveredtheir mobility over time after rinsing and transferring them in water, showing a nematostatic activity of tannins. In the potexperiment, tannins, as aqueous solutions at rates from 0.30 to 1.50 g L−1, were applied to soil at three different applicationtimes (1: only at transplant; 2: at transplant, two weeks after transplant and repeated every seven days; 3: at transplant andtwo weeks later). The activity of tannins was compared to treated and untreated controls. Tested rates mostly repeated wereeffective to control nematode attack in comparison to untreated control. The height of treated plants was not significantlyinfluenced by the different applied rates of tannins, whereas nematode population density and root galling index wereaffected by repeated application times. No visual symptoms of phytotoxicity were detected. The use of SaviotaN®appearspromising for the control of M. incognitain sustainable agriculture of short-term crops and/or when nematode populationdensities are low and as a supplement to other chemical treatments

Trichoderma-based products and their widespread use in agriculture

Governing bodies throughout the world, particularly in Europe, are now implementing legislative mandates with the objective of decreasing dependence on pesticides in agriculture to increase consumer and environmental safety. In order to reduce the risks associated with pesticide applications and reduce dependency on their use, Directives will promote low pesticide-input by implementing integrated pest management (IPM), and provide the means to establish the necessary conditions and measures to employ these practices, as well as to ensure security of commercial products. One approach includes the use of biological control agents and their products as alternatives to synthetic agro-chemicals. Trichoderma spp. are widely studied fungi and are among the most commonly used microbial biological control agents (MBCAs) in agriculture. They are presently marketed as bio-pesticides, biofertilizers, growth enhancers and stimulants of natural resistance. The efficacy of this fungus can be attributed to their ability to protect plants, enhance vegetative growth and contain pathogen populations under numerous agricultural conditions, as well as to act as soil amendments/inoculants for improvement of nutrient ability, decomposition and biodegradation. The living fungal spores (active substance) are incorporated in various formulations, both traditional and innovative, for applications as foliar sprays, pre-planting applications to seed or propagation material, post-pruning treatments, incorporation in the soil during seeding or transplant, watering by irrigation or applied as a root drench or dip. Trichoderma-based preparations are marketed worldwide and used for crop protection of various plant pathogens or increase the plant growth and productivity in diverse cultivated environments such as fields, greenhouses, nurseries; in the production of a variety of horticultural, fruits, trees and ornamental crops. A survey was conducted of Trichoderma-containing products found on the international market to obtain an overall perspective of the: 1) geographical distribution, 2) product composition and identity of Trichoderma species selected, 3) contents combined with Trichoderma in the products - other microbial species or substances in the mix, 4) number of products available globally and geographically, 5) number of products registered or having use specifications, 6) product formulations and applications, 7) manufacturer claims - target use, target pests, product type and effects of applications. The largest distribution of Trichoderma bioproducts is found in Asia, succeeded by Europe, South- Central America and North America. The majority of the labels indicated fungicidal properties, but only 38% of the marketed merchandise are registered. Ten Trichoderma species are specifically indicated, but many labels indicate a generic Trichoderma sp. or spp. mix in the list of ingredients. The most common formulation is a wettable powder, followed by granules. Generally, Trichoderma are applied to the seed or propagation material at the time of planting, then the secondary use is during plant development. On the whole, the target use is for the control of soilborne fungal pathogens such as Rhizoctonia, Pythium and Sclerotinia, and a few foliar pathogens such as Botrytis and Alternaria; whereas the minor use indication is for plant growth promotion. The use of Trichoderma-based biological products will have an important role in agricultural production of the future, in light of changing worldwide perspectives by consumers and governing bodies

Trichoderma and its secondary metabolites improve yield and quality of grapes

Trichoderma is one of the most studied and applied fungal biocontrol agents. The benefits of these microorganisms to the plant include: suppression of pathogens, growth promotion, enhanced nutrient availability and induction of resistance. The biological activity is related to the variety of metabolites that they produce. These metabolites have been found to directly inhibit the pathogens, increase disease resistance and enhance plant growth. In this study, we have examined the effect of two Trichoderma strains and their secondary metabolites on Vitis vinifera in terms of induction of disease resistance, plant growth promotion and increase of polyphenols or antioxidant activity in the grapes. Applications of T. harzianum M10 or T. atroviride P1, as well as their respective major secondary metabolites, harzianic acid (HA) and 6-pentyl-a-pyrone (6PP), have been conducted in greenhouse by foliar spray or drenching. The treatments suppressed the development of powdery mildew caused by Uncinula necator. In a field experiment, a spore suspension of T. harzianum strain T22 or a 6PP solution was applied until fruit harvest. The results indicated that both T. harzianum T22 and 6PP are able to improve crop yield and increase the total amount of polyphenols and antioxidant activity in the grapes. The effects of the isolated natural compounds were comparable with those obtained by using the living fungus

Trichoderma secondary metabolites active on plants and fungal pathogens

Beneficial microbes typically produce bioactive molecules that can affect the interactions of plants with their pathogens. Many secondary metabolites may also have antibiotic properties, which enable the producing microbe to inhibit and/or kill other microorganisms i.e. competing for a nutritional niche. Indeed, some of these compounds have been found to play an important role in the biocontrol of plant diseases by various beneficial microbes used world-wide for crop protection and bio-fertilization. In addition to direct toxic activity against plant pathogens, biocontrol-related metabolites may also increase disease resistance by triggering systemic plant defence activity, and/or enhance root and shoot growth. Fungi belonging to the Trichoderma genus are well known producers of secondary metabolites with a direct activity against phytopathogens and compounds that substantially affect the metabolism of the plant. The widescale application of selected metabolites to induce host resistance and/or to promote crop yield may become a reality in the near future and represents a powerful tool for the implementation of IPM strategies

Multiple roles and effects of a novel Trichoderma hydrophobin

Fungi belonging to the genus Trichoderma are among the most active and ecologically successful microbes found in natural environments, as they are able to use a variety of substrates and affect the growth of other microbes and virtually any plant species. We isolated and characterized a novel type II hydrophobin secreted by the biocontrol strain MK1 of Trichoderma longibrachiatum. The corresponding gene (Hytlo1) has a multiple role in the Trichoderma-plant-pathogen three-way interaction, while the purified protein displayed a direct antifungal as well as a MAMP and a plant growth promotion (PGP) activity. Leaf infiltration with the hydrophobin systemically increased resistance to pathogens and activated defence-related responses involving ROS, SOD, oxylipins, phytoalexins and PR-proteins formation or activity. The hydrophobin was found to enhance development of a variety of plants when applied at very low doses. It particularly stimulated root formation and growth, as demonstrated also by transient expression of the encoding gene in tobacco and tomato. Targeted knock-out of Hytlo1 significantly reduced both antagonistic and PGP effect of the WT strain. We conclude that this protein represents a clear example of a molecular factor developed by Trichoderma to establish a mutually beneficial interaction with the colonized plant

Modulation of Tomato Response to Rhizoctonia solani by Trichoderma harzianum and Its Secondary Metabolite Harzianic Acid

The present study investigated the transcriptomic and metabolomic changes elicited in tomato plants (Solanum lycopersicum cv. Micro-Tom) following treatments with the biocontrol agent Trichoderma harzianum strain M10 or its purified secondary metabolite harzianic acid (HA), in the presence or the absence of the soil-borne pathogen Rhizoctonia solani. Transcriptomic analysis allowed the identification of differentially expressed genes (DEGs) that play a pivotal role in resistance to biotic stress. Overall, the results support the ability of T. harzianum M10 to activate defense responses in infected tomato plants. An induction of hormone-mediated signaling was observed, as shown by the up-regulation of genes involved in the ethylene and jasmonate (ET/JA) and salicylic acid (SA)-mediated signaling pathways. Further, the protective action of T. harzianum on the host was revealed by the over-expression of genes able to detoxify cells from reactive oxygen species (ROS). On the other hand, HA treatment also stimulated tomato response to the pathogen by inducing the expression of several genes involved in defense response (including protease inhibitors, resistance proteins like CC-NBS-LRR) and hormone interplay. The accumulation of steroidal glycoalkaloids in the plant after treatments with either T. harzianum or HA, as determined by metabolomic analysis, confirmed the complexity of the plant response to beneficial microbes, demonstrating that these microorganisms are also capable of activating the chemical defenses

PRODUZIONE DI NUOVI CEPPI DI TRICHODERMA SPP. CON CARATTERISTICHE BENEFICHE MULTIPLE

L’analisi Microarray è stata utilizzata per studiare l’espressione genica in pomodoro (Solanum lycopersicum cv Microtom) durante l’interazione con il T. harzianum ceppo M10 o il suo metabolita secondario acido harzianico (HA), in presenza-assenza del patogeno R. solani. I diversi trattamenti effettuati sulle piante con l’agente di biocontrollo, il metabolita secondario e il patogeno hanno permesso di individuare cambiamenti nell’espressione genica sia nell’interazione a due che a tre componenti. Nel caso dell’interazione bipartita, il numero dei DEG (Differentially Expressed Genes) è risultato maggiore nel confronto Pianta-M10 vs Controllo; difatti sono stati identificati 1308 DEGs (1214 up-regulated e 94 down-regulated), mentre nel confronto Pianta-HA vs Controllo è stato annotato un unico gene differenzialmente espresso, risultato sotto regolato. In seguito al trattamento con T. harzianum ceppo M10, la pianta ha mostrato una sovra-espressione di geni coinvolti in processi di glicolisi, TCA e fotosintesi, oltre alla sovra-espressione di geni quali estensina, XTH, NAC e COP9 che sono generalmente associati ai meccanismi di crescita e sviluppo della pianta, suggerendo quindi una capacità del fungo di stimolare tali processi. Lo stesso trattamento ha mostrato una sovra-espressione di alcuni geni codificanti per chitinasi, ovvero enzimi classificati come proteine di patogenesi (PR). Nell’interazione tripartita, in presenza del patogeno Rhizoctonia, il numero di geni differenzialmente espressi è risultato maggiore rispetto all’interazione bipartita. Molti di questi DEG sono risultati coinvolti in molteplici risposte di difesa. Analizzando il confronto pianta infetta-M10 vs Pianta infetta, sono stati individuati 1218 DEG, dei quali 661 sovra-espressi e 557 sotto-espressi; nel trattamento pianta infetta-HA vs Pianta infetta sono stati identificati 2507 DEGs, di cui 1517 sovra-espressi e 990 sotto-espressi. Nel confronto tra i trattamenti Pianta-HA-R. solani vs Pianta-M10-R. solani non sono stati identificati geni differenzialmente espressi. I trattamenti con Trichoderma sembrano avere un’azione protettiva sulla pianta ospite mostrata dalla sovra-espressione di geni in grado di detossificare i composti ROS che, se accumulati, possono danneggiare ed essere tossici nelle cellule vegetali. In accordo con i risultati dell’interazione bipartita Pianta-Trichoderma, sono stati identificati geni codificanti per le proteine “Multi bridgingfactor 1”, ER-24 e EIN3 che indicano la presenza dell’ormone etilene e, la concomitante attivazione della via dell’acido jasmonico, suggerendo quindi un effetto sui meccanismi di resistenza (ISR). La presenza di Trichoderma, ha un effetto sull’espressione di geni coinvolti nel processo di difesa della pianta, sia essa sana (priming), sia soggetta all’attacco di un patogeno. Questo studio ha fornito indicazioni importanti sull’effetto positivo che l’agente di biocontrollo e il suo metabolita esplicano sulla promozione della crescita e attivazione delle risposte di difesa in pomodoro. Brachypodium distachyon è una pianta che appartiene alla classe delle monocotiledoni, famiglia Poaceae, che comprende le colture di maggior interesse commerciale per la produzione agricola, come i cereali: frumento, mais e riso. B. distachyon viene proposta come coltura alternativa a Panicum virgatum, per la produzione di biocarburanti. Gli studi dell’interazione B. distachyon-microrganismi benefici potrebbero fornire indicazioni importanti per una produzione sostenibile di bioenergia grazie ad un aumento della biomassa vegetale e della resistenza a stress. Poiché non sono ancora noti in letteratura studi che riguardano il sistema Brachypodium-Trichoderma ed i risultati possono variare enormemente a seconda della specie vegetale, del ceppo o dei parametri utilizzati nei trattamenti, una prima fase del lavoro ha riguardato la definizione delle condizioni ottimali per lo svolgimento delle successive sperimentazioni che hanno riguardato il trattamento preliminare dei semi, la scelta del tipo di suolo su cui effettuare i saggi, la tipologia di accessione da utilizzare. Analogamente sono state selezionate diverse specie di Trichoderma, isolate da molteplico formulati commerciali registrati come biofungicidi e/o biofertilizzanti. Da uno screening preliminare sono stati selezionati 3 ceppi (M10, Myc1 e RS2, tutti appartenenti alla specie T. harzianum) che hanno evidenziato la migliore attività in termini di incremento della crescita e di biomassa vegetale in B. dystachion. Dal momento che, per la produzione di bioenergia è necessario impiegare la parte epigea della pianta, la selezione dei ceppi e le analisi che sono seguite si sono concentrate soprattutto sui trattamenti in grado di determinare un maggior incremento del peso secco dell’apparato fogliare delle piante trattate. Una prima considerazione emersa dall’osservazione dei risultati ottenuti in questo lavoro è che la specie T. harzianum ha mostrato un’attività ospite-specifica nei confronti di B. dystachion rispetto alle altre 3 specie di Trichoderma impiegate. Questa ipotesi trova conferma in letteratura dove viene riportato che esiste una componente genetica della pianta che può influenzare l’effetto del ceppo (Shoresh e Harman, 2008). Per quanto riguarda le modalità di trattamento dei semi con i ceppi selezionati, la sola concia ha determinato un notevole incremento del peso fresco delle piante trattate rispetto al controllo. Inoltre non è stato osservato alcun effetto di promozione della crescita dovuto al trattamento dei semi con miscele di spore sterilizzate (e quindi non più vitali), indicando che l’effetto positivo sulle piante è dovuto al BCA vivente. L’isolamento e la determinazione della concentrazione degli endofiti presenti a livello radicale ha inoltre mostrato un buon livello di colonizzazione da parte dei ceppi di Trichoderma applicati sulla pianta ospite. Ad ogni modo, quando al trattamento al seme seguivano trattamenti settimanali al suolo con le sospensioni di spore dei ceppi selezionati, gli incrementi del peso fresco delle piante rispetto al controllo sono stati di gran lunga maggiori. Le performance dei ceppi sono state monitorate a cadenza settimanale e in diversi casi sono stati riscontrati incrementi di peso superiori al 100% rispetto ai controlli. Particolarmente interessante è risultato l’incremento del peso secco nelle piante dopo due settimane dal trattamento con il ceppo M10 rispetto al controllo (+124% con la sola concia del seme, e +225% con la concia seguita dagli inoculi settimanali). Questi dati trovano conferma negli innumerevoli studi presenti in letteratura circa la capacità di Trichoderma di migliorare la crescita delle piante attraverso una serie di meccanismi (incremento della solubilizzazione dei nutrienti presenti nel suolo e della capacità della pianta di assorbirli, stimolazione della crescita dell’apparato radicale, cambiamento della composizione della microflora radicale, etc.). In conclusione i risultati ottenuti dallo studio dell’interazione Brachypodium-Trichoderma dimostrano come l’utilizzo di ceppi selezionati di questi funghi benefici possa rappresentare una valida strategia da utilizzare per promuovere la crescita e lo sviluppo di questa coltura utilizzata per produzione di bioenergia. Con la stessa finalità è stata utilizzata una strategia innovativa basata sull’ottenimento di nuovi ceppi ibridi di Trichoderma a partire da parentali selezionati sulla base di riconosciute proprietà benefiche nei confronti delle piante. Nello specifico sono stati selezionati tre BCA appartenenti alle specie T. harzianum (ceppo M10), T. longibrachiatum (ceppo MK1) e T. atroviride (ceppo P1), dotati di capacità di: antagonismo contro patogeni terricoli e fogliari; produzione di metaboliti secondari attivi; promozione della crescita delle piante; ed induzione di resistenza sistemica. Gli ibridi ottenuti mediante la fusione dei protoplasti di coppie di ceppi selezionati hanno mostrato in diversi casi un potenziamento della stimolazione della crescita delle piante o dell’attività di biocontrollo di patogeni rispetto ai ceppi parentali. I risultati più significativi si sono registrati in termini di allungamento radicale e crescita dell’apparato epigeo delle piante di pomodoro, mentre solo un ibrido selezionato ha determinato un anticipo della germinazione dei semi risultato statisticamente significativo rispetto ai parentali e al controllo. Tutti i ceppi impiegati hanno mostrato capacità di biocontrollo nei confronti del patogeno terricolo S. rolfsii e di quello fogliare B. cinerea e in taluni casi la capacità di inibizione dell’infezione dei ceppi derivati da fusione è risultata superiore a quella dei rispettivi parentali. Questo approccio impiegato per la produzione di ceppi ibridi rappresenta un’assoluta novità poiché non prevede la mutagenesi dei ceppi impiegati come parentali ed è in grado di combinare diversi caratteri desiderabili in un unico individuo

Early Detection of Wild Rocket Tracheofusariosis Using Hyperspectral Image-Based Machine Learning

Fusarium oxysporum f. sp. raphani is responsible for wilting wild rocket (Diplotaxis tenuifolia L. [D.C.]). A machine learning model based on hyperspectral data was constructed to monitor disease progression. Thus, pathogenesis after artificial inoculation was monitored over a 15-day period by symptom assessment, qPCR pathogen quantification, and hyperspectral imaging. The host colonization by a pathogen evolved accordingly with symptoms as confirmed by qPCR. Spectral data showed differences as early as 5-day post infection and 12 hypespectral vegetation indices were selected to follow disease development. The hyperspectral dataset was used to feed the XGBoost machine learning algorithm with the aim of developing a model that discriminates between healthy and infected plants during the time. The multiple cross-prediction strategy of the pixel-level models was able to detect hyperspectral disease profiles with an average accuracy of 0.8. For healthy pixel detection, the mean Precision value was 0.78, the Recall was 0.88, and the F1 Score was 0.82. For infected pixel detection, the average evaluation metrics were Precision: 0.73, Recall: 0.57, and F1 Score: 0.63. Machine learning paves the way for automatic early detection of infected plants, even a few days after infection