Garlic dry rot: a comprehensive study from field to fork on casual agents and disease management

L’aglio è coltivato a livello mondiale nelle regioni temperate, secondo la FAO nel 2016 1.5 milioni di ettari sono stati destinati a questa coltura. A partire dal 2002 Fusarium proliferatum è stato segnalato come principale agente causale del marciume secco dell’aglio in post raccolta. I sintomi sono identificabili come macchie necrotiche sui bulbilli e in presenza di forti infezioni è possibile osservare la presenza di micelio bianco. F. proliferatum è un patogeno in grado di produrre Fumonisina B1 e B2, micotossine che potrebbero accumularsi all’interno dei bulbi ed essere tossiche per il consumatore. In Italia nel 2012 il 30% del raccolto è stato perso a causa di questo patogeno. Essendo la Fusariosi una malattia emergente a livello mondiale in letteratura si trovano ancora scarse informazioni sul suo sviluppo a livello di campo e sulle strategie di contenimento. 1. Allo scopo di verificare la quantità di inoculo fungino presente nell’ambiente si è proceduto all’analisi dei suoli in presemina con specifica attenzione alla quantificazione e all’identificazione delle specie fungine presenti. A tale scopo è stata eseguita: la conta delle Unità Formanti Colonia per grammo di terreno (UFC/g) su terreni specifici per l’isolamento del genere Fusarium spp. per grammo di terreno. Le identificazioni sono state eseguite al microscopio ottico e confermate successivamente con metodi molecolari. 2. Per seguire l’avanzamento della malattia durante la stagione colturale, invece, si è proceduto al campionamento in tre fasi fenologiche (inizio formazione dei bulbilli BBCH 15, ingrossamento dei bulbilli BBCH 45, maturazione di raccolta BBCH 49) con caratterizzazione dei sintomi, isolamento e riconoscimento dei funghi associati al marciume. 3. Per verificare la correlazione tra andamento meteo e incidenza delle specie fungine associate al marciume secco, sono stati raccolti i dati di meteorologici relativi al totale delle piogge, ai gradi giorno, all’umidità relativa media e alla temperatura media nei quadrati corrispondenti alle aziende agricole oggetto di studio. I dati sono stati correlati attraverso il coefficiente di correlazione di Pearson con i valori di gravità e incidenza della malattia stimati a fine stagione colturale. 4. Per verificare l’insorgenza dalla malattia nella fase di post raccolta si è proceduto con campionamenti di bulbi in conservazione, posa in piastra di bulbilli sintomatici e asintomatici e calcolo dell’incidenza delle specie fungine associate ai sintomi del marciume. 5. Al fine di verificare la possibile presenza di fumonisine nei campioni analizzati durante la stagione colturale e nel post raccolta, si è proceduto all’analisi attraverso HPLC di estratti di aglio. 6. Per individuare possibili strategie di controllo della malattia durante la stagione colturale sono stati eseguiti test di efficacia di prodotti chimici e biologici in vitro e in campo. I prodotti chimici sono stati provati su PDA modificato inoculato centralmente con F. proliferatum, mentre per gli agenti di biocontrollo sono state allestite prove di coltura duale. La prova in campo, invece, è stata eseguita all’interno di un campo sperimentale a strip plot. L’aglio delle diverse tesi è stato conservato in cella frigorifera per 9 mesi, per valutare la persistenza dei prodotti utilizzati alla concia. I risultati ottenuti hanno dimostrato che F. proliferatum e F. oxysporum sono le specie maggiormente associate al marciume dell’aglio durante la stagione colturale. L’andamento delle due specie è complementare e varia a seconda dell’andamento meteorologico della stagione colturale. F. proliferatum è correlato positivamente con l’aumento della temperatura e delle piogge, mentre F. oxysporum sembra prevalere nelle stagioni meno piovose ed ha mostrato correlazione positiva con la gravità dei sintomi rilevati in campo sulle corone. La carica micotica di Fusarium nel terreno rimane costante negli anni di analisi, facendo presupporre un maggiore ruolo del seme nella trasmissione della malattia. Per quanto riguarda il post raccolta, invece, F. proliferatum risulta la specie isolata con maggiore frequenza dai bulbilli e si correla positivamente ai sintomi rilevati sugli spicchi, confermando il suo ruolo come agente causale del marciume secco durante lo stoccaggio. F. oxysporum, invece, colonizza in prevalenza le radici e la parte basale della pianta dividendo il patosistema in due subsistemi: F. proliferatum-bulbi; F. oxysporum-radici. F. proliferatum è stato isolato anche dagli spicchi asintomatici con frequenza del 25%, ed è stato possibile rilevare la presenza di fumonisine con l’avanzare del tempo di stoccaggio in cella. Essendo il fungo presente anche sugli spicchi asintomatici maggiori studi saranno necessari per garantire la sicurezza dei consumatori. Infine, dalle prove di concia in campo è emerso che il principio attivo Tebuconazolo, riduce la comparsa dei sintomi da Fusarium, ma non in modo risolutivo. Ciononostante, una volta che il prodotto viene riportato a temperatura ambiente dopo lo stoccaggio in cella refrigerata, l’incidenza di F. proliferatum aumenta nuovamente con possibilità di sviluppo di danni al prodotto da commercializzare.Since 2002, Fusarium proliferatum has been reported as the main causal agent of garlic dry rot during the postharvest stage, but information on the development of the disease throughout the production chain was nearly absent. Dry rot has caused huge economic losses in the past few years (up to 30 % of the yield), symptoms are visible on bulbs during storage as necrotic spots and in the most severe attacks, white mycelium may become visible on cloves. Few pest management strategies were tested in the recent past, but none were satisfactory. Due to the economic effect that this pathogen can have on local productions, the thesis aimed to deeply investigate the pathosystem with a field to fork approach and to test new strategies to control fungal infections. First of all, the work focused on garlic (Allium sativum L.) cropping season, intending to clarify the role of F. proliferatum in bulb infection as well as the impact of crop growing conditions on the development of the pathogen. A 3-year study was conducted in Piacenza (northern Italy) by sampling six garlic farms with different dry rot history (three highly contaminated and three low contaminated). Soil samples were recovered at sowing time for the counting of fungal colony-forming units (CFU). Plant samples were collected at three relevant growth stages, from April to July, for which disease severity assessment and fungi isolations were performed. Fusarium was the most frequently isolated genus, and F. proliferatum and F. oxysporum the dominant species during the garlic cropping season. F. oxysporum was dominant in the first year of the study, but F. proliferatum registered the highest incidence in all the farms tested. F. oxysporum incidence was correlated with dry weather, whereas F. proliferatum was enhanced in rainy years. To conclude, F. proliferatum is confirmed to be associated with garlic bulbs, even at crop’s early growth stages and symptoms are visible mainly on roots and basal plates at the field stage, related to F. oxysporum. Then, the focus was made in detecting the presence of F. proliferatum on garlic bulbs during prolonged storage, and to identify other fungal species associated with garlic dry rot. Moreover, fumonisin contamination in symptomatic and asymptomatic cloves were detected. Samples of 100 plants were collected over three production seasons in six farms located in Northern Italy at three-time points (at harvest, processing, and 6 months storage at –4° C). Results obtained lead to think that Fusarium–garlic pathosystem is split into two parts: basal plate/root and bulb. F. proliferatum had the highest incidence in infected bulbs and was confirmed as the causal agent of postharvest dry rot in garlic (mean incidence: 35.4%). F. oxysporum co-occurred with F. proliferatum but symptoms were visible only on basal plate/root. Dry rot incidence slightly increased during cold storage (from 14.6% at processing to 18.4% at 6-month storage); although, F. proliferatum incidence was stable during cold storage, fumonisin were produced from harvest through storage. Cloves showing symptoms were more contaminated compared to those asymptomatic, both by the fungus (mean incidence 39% vs 25.3%) and the toxin (287.0 vs 24.4 µg kg-1). Therefore, cold storage limits garlic dry rot, but health concerns related to fumonisin should be seriously considered. Regarding disease management, garlic crop is commonly propagated by plant parts (cloves). To protect garlic crop from early growth stages it is important to find commercial products able to control the pathogen growth on seedlings. The experiment aimed to test in vitro and in vivo the efficacy of triazoles and biocontrol agents (BCAs) against F. proliferatum and F. oxysporum. In in vitro trials, the best performance was achieved by propiconazole+prochloraz (100%), followed by tebuconazole (88.9%). BCAs were less effective but still showed great capacity to control the pathogen with maximum growth inhibition of 80% (Trichoderma harzianum +T. gamsii). In both cases, temperature influenced the capacity to control the pathogen with minimum effect at 25°C compared to lower temperatures. In vivo bacterial BCAs showed a similar capacity to control Fusaria compared to chemical products (mean of severity index 18.6% and 11.7%, respectively) and did not show side effects on root length. In vitro and in vivo results are comparable, except for Trichoderma, with the worst performances in terms of disease severity on plants. Finally, a field trial was designed to verify the efficacy of chemical and biological active ingredients as seed coating both at crop stage and postharvest, simulating the entire production chain, by taking into account visible symptoms and incidence of fungi. All products tested reduced the severity of symptoms on basal plates at the field stage, but none of them was able to reduce Fusarium incidence. A postharvest analysis conducted on bulbs demonstrated the efficacy of Tebuconazole, B. subtilis, and Trichoderma+B. subtilis in reducing the number of cloves showing symptoms per bulb (mean 34.3% vs control 45.8%). Moreover, Tebuconazole was able to reduce the incidence of F. proliferatum by 48% with respect to untreated control. The trial highlighted also that the incidence of F. proliferatum increased by 37% when garlic bulbs were kept for 15 days at room temperature simulating storage at consumers houses. Results obtained in the trial are promising and seed coating had a positive effect on garlic dry rot postharvest; although further studies are needed to test the persistence of seed coating treatments after prolonged storage period, especially when the product is kept outside cold chambers

Chemical and biological control of Fusarium species involved in garlic dry rot at early crop stages

AbstractThe aim of the study was to test in vitro and in vivo the efficacy of triazoles and biocontrol agents (BCAs) against Fusarium proliferatum and F. oxysporum, the former signaled as the main causal agent of garlic dry rot and the latter also involved. In vitro trials were organized using potato dextrose agar with added chemicals or BCAs inoculated with selected F. proliferatum and F. oxysporum. Garlic cloves were dipped before sowing in suspensions prepared with the fungicides showing the best performances in vitro; then they were dipped in Fusaria suspension before sowing. In in vitro trials, the maximum Fusaria growth inhibition was performed by Propiconazole + Prochloraz (100%), followed by Tebuconazole (88.9%). BCAs showed great capacity to control Fusaria, with a maximum growth inhibition of 80% (Trichoderma harzianum + T. gamsii). In vivo bacterial BCAs showed a similar capacity to control F. proliferatum and F. oxysporum compared to chemical products (mean of severity index 18.6% and 11.7%, respectively). In vivo results confirmed the in vitro performances, except for Trichoderma, which had the worst performances in vivo. Therefore, the results are preliminary but promising for future field application

Efficacy of chemical and biological spray seed treatments in preventing garlic dry rot

Garlic dry rot caused by Fusarium proliferatum is an emerging postharvest disease that has resulted in severe economic losses, necessitating design and implementation of efficient disease control strategies. Sanitation of planting cloves is critical for preventing garlic dry rot. This study evaluated the efficacy of commercial chemicals and biocontrol agents, applied at planting as spray treatments, for reducing disease severity and the occurrence of Fusarium spp. in garlic, from the field stage then through 9 months of postharvest storage. Tebuconazole was the most effective for reducing disease severity, giving 26.5% reduction of basal plate rots and 44% reduction of bulb rots, and 33.4% reduction in visible symptoms on cloves relative to the untreated controls. Comparable results were obtained by applying B. subtilis and S. griseoviridis. However, none of the active ingredients tested in this study reduced the incidence of F. oxysporum and F. proliferatum on basal plates, although tebuconazole reduced the postharvest incidence of F. proliferatum on cloves by nearly 50%. Incidence of F. proliferatum increased by 37% in bulbs transferred from storage to room temperature (25°C) for 15 days, simulating storage in consumers' homes. These results demonstrate that spray seed clove treatments have inhibitory effects on postharvest garlic dry rot, although further research is required to determine the persistence of these treatments during prolonged storage, especially without low temperatures

Fungi Associated with Garlic During the Cropping Season, with Focus on Fusarium proliferatum and F. oxysporum

Fusarium proliferatum has been reported as the main causal agent of garlic dry rot during the postharvest stage, but information on this fungus during the crop growth stage is lacking. We focused on the cropping season of garlic (Allium sativum L.) in the field, until its harvest, with the aim of clarifying the role of F. proliferatum in bulb infection as well as the impact of crop growing conditions on pathogen-plant interaction. Studies were conducted in Piacenza (northern Italy) for three seasons from 2016 to 2019. Six garlic farms were sampled. A different field was sampled every year. Soil samples were recovered at sowing time for the counting of fungal colony forming units (CFU). Plant samples were collected at three growth stages, from BBCH 15 (fifth leaf visible) to BBCH 49 (ripening), for which disease severity assessment and fungi isolations were performed. Fusarium was the most frequently isolated genus, of which F. proliferatum and F. oxysporum were the dominant species. F. proliferatum registered the highest incidence in all the farms tested, but F. oxysporum was dominant in the first year of the study. F. oxysporum incidence was correlated with dry weather, whereas F. proliferatum was correlated with rainy weather. In conclusion, our result confirms the association of F. proliferatum with garlic bulbs from the crop's early growth stages, suggesting potential seed transmission as a source of this fungal pathogen. Further studies should investigate the link between fusaria occurrence in the field and dry rot outbreaks occurring postharvest and during storage of garlic

Comparison of different physical methods and preservatives for control of Fusarium proliferatum rot in garlic

Dry rot is an emerging issue for garlic production worldwide and Fusarium proliferatum is its major causal agent. Since the disease is seed-transmitted, sowing healthy cloves is crucial. In this study, some disinfection strategies were tested on garlic seeds, including steam, dry heat, chemical disinfectants and gaseous ozone (O3). Steam reduced the Colony Forming Units·g−1 (CFUs·g−1) by up to 92% in garlic seeds, but, at the same time, it affected their germination (−36%). Similarly, hydrogen peroxide (H2O2) and peracetic acid (C2H4O3) reduced the CFUs·g−1 by up to 83%; however, these methods also severely impaired germination (−40%). Dry heat did not negatively impact germination, but fungal contamination was not significantly reduced. The most promising strategy was gaseous O3 treatment; it decreased CFUs·g−1 by up to 96%, without causing any reduction of germination. The treatments applied were partially effective because the fungus is predominantly located in the outer layer of the seed, although it is also found in the inner portions. Some of these treatments can contribute to garlic protection from seed-borne pathogens and possibly reduce the occurrence of garlic dry rot

In vitro efficacy tests against Fusarium spp. causing garlic dry rot

Garlic is cultivated worldwide and, according to FAOSTAT, in 2016 1.5 million hectares were destined to this culture. Starting from 2002, Fusarium proliferatum was signalled as the main causal agent of garlic dry rot during storage, but with infections starting during the growing season. Seed sanitation seems crucial to prevent pathogen spread. In the context of preparing guidelines for farmers to prevent/control dry rot in field and during storage, this study was designed. Fusarium proliferatum (MPVPG29) recovered from garlic during the 2017 growing season was used in in vitro trials. Six fungicides, all including triazoles, alone or in mixture, and 4 competitor/antagonist biocontrol agents (BCA; Trichoderma gamsii, Fusarium oxysporum IF23, Bacillus subtilis, Streptomyces griseoviridis K61) were considered. Spiked media for chemicals (4 concentrations (1-1000 ppm) and dual cultures for BCAs were used. Inoculated dishes were incubated at 3 temperatures (10-15-25\ub0 C) and the percentage of growth inhibition was recorded after 7-14-21 days. All chemical products were able to stop fungal growth at 100 ppm concentrations and Procloraz+Propiconazole showed a 94% reduction followed by Tebuconazole (57% reduction) even at 1 ppm. Regarding BCAs, T. gamsii was the most effective in reducing F. proliferatum growth (mean reduction=84%), followed by S. griseoviridis (mean reduction=60%). Results are very promising and a field trial is ongoing to verify the performances of BCAs in garlic crops

FIELD INVESTIGATION ON GARLIC DRY ROT

Fusarium proliferatum was signalled worldwide since 2002 as the main causal agent of garlic dry rot. According to literature, other Fusarium spp and nematodes (Ditylenchus dipsaci) could contribute to the disease. Moreover, white varieties are reported as more susceptible compared to red ones. Dry rot is considered a postharvest disease, but relevant incidence of symptomatic bulbs at harvest were also reported. The aim of this study was to investigate infection time and agent involved in garlic dry rot from field to table. Field sampling was organised in Piacenza province (north Italy), area of production of white garlic (PGI). Six field units were selected for sampling, 3 of them with an history of relevant dry rot. Soil was sampled before sowing. Garlic plants were collected in three growth stages: BBCH 15 (5th leaf clearly visible), BBCH 45 (50% of the expected bulb diameter reached), BBCH 49 (Leaves dead, bulb top dry; growth complete). With soil, serial dilutions and colony forming units count (CFU) were performed so as nematode counting. Direct isolation from symptomatic and asymptomatic plants was managed, so as the identification at species level for a selected set of fungal strains. PCR was applied to confirm fungi and nematode identification. Ditylenchus dipsaci was not detected in soil in autumn. Regarding fungi, the largely dominant species were F.proliferatum and F.oxysporum, isolated since BBCH 15, increasing in incidence from early growth stages to harvest. F. proliferatum seems confirmed as the candidate most relevant causal agent of dry rot, infecting bulbs early in field

Abutilon theophrasti’s Root Associated Microorganisms Support Hydroxylation-dependent Benzoxazolinone Detoxification and Degradation

Abutilon theophrasti is one of the most problematic weeds worldwide. Rye mulches, rich in benzoxazinoids, could not suppress Abutilon (Herbiseeds) due to its avoidance of high BOA/BOA detoxification product accumulation and phytotoxic 2-aminophenoxazinone production. Since Abutilon theophrasti seed coats harbour a variety of fungi and bacteria, a participation of those organisms in BOA degradation was assumed. The avoidance of high detoxification product/BOA accumulation in the roots was correlated with the presence of the Zygomycete Actinomucor elegans, which is known to colonize Abutilon seed coats. The microorganism(s) produced plant growth promoting compounds, whereby tryptophan excretion was stimulated by BOA. In Fenton reactions with BOA, tryptophan accelerates the destruction of the allelochemical via hydroxylated BOA molecules, when used in defined ratios. Destruction of BOA via hydroxylated molecules presents a new pathway for BOA degradation without accumulation of 2-aminophenoxazinone. During this process tryptophan is also degraded. Applications of high concentrations of tryptophan led, however, to a higher, channelled synthesis of BOA-6-O-glucoside in the roots, probably to slow down oxygen consuming Fenton reactions. From all possible hydroxylated BOA molecules, BOA-6-OH is the best substrate for Oglucosylation. Incubations in presence of the mono-oxygenase effector 2- bromo-4´-nitroacetophenone revealed that those enzymes are mainly responsible for BOA-6-OH synthesis and only a minor part results from Fenton reactions. The study elucidates the important role of plant-microorganism associations in the survival of allelopathic attacks

Comparison of Different Physical Methods and Preservatives for Control of Fusarium proliferatum Rot in Garlic

Dry rot is an emerging issue for garlic production worldwide and Fusarium proliferatum is its major causal agent. Since the disease is seed-transmitted, sowing healthy cloves is crucial. In this study, some disinfection strategies were tested on garlic seeds, including steam, dry heat, chemical disinfectants and gaseous ozone (O3). Steam reduced the Colony Forming Units·g−1 (CFUs·g−1) by up to 92% in garlic seeds, but, at the same time, it affected their germination (−36%). Similarly, hydrogen peroxide (H2O2) and peracetic acid (C2H4O3) reduced the CFUs·g−1 by up to 83%; however, these methods also severely impaired germination (−40%). Dry heat did not negatively impact germination, but fungal contamination was not significantly reduced. The most promising strategy was gaseous O3 treatment; it decreased CFUs·g−1 by up to 96%, without causing any reduction of germination. The treatments applied were partially effective because the fungus is predominantly located in the outer layer of the seed, although it is also found in the inner portions. Some of these treatments can contribute to garlic protection from seed-borne pathogens and possibly reduce the occurrence of garlic dry rot

Abutilon theophrasti's root associated microorganisms support hydroxylation-dependent benzoxalinone detoxification and degradation

The study elucidate the important role of plant-microorganism associations in the survival of allelopathic attacks