New Antifungal Compound, 6-Pentyl- α -Pyrone, against the Maize Late Wilt Pathogen, Magnaporthiopsis maydis

: 6-pentyl- α -pyrone (6-PP) is a powerful Magnaporthiopsis maydis antifungal compound, recently discovered when the potent growth medium of Trichoderma asperellum was analyzed. Despite its high potential in plate assay, it was not inspected for plant treatment prevention. Late wilt disease, caused by the fungus M. maydis , threatens commercial maize production in high-risk areas. Thus, the search for control options against the pathogen is one of the top priorities in Israel, Egypt, and other countries. Disease-resistant maize genotypes can reduce the damages. Yet, aggressive variants of the fungus can overcome host resistance. The current study aimed at inspecting T. asperellum and its secreted metabolite, pure 6-PP, against the pathogen in plants over a full growth period. First, adding T. asperellum directly to seeds with sowing provides signiﬁcant protection to sprouts (up to 42 days) in a growth room, with more than two-fold growth promotion and reduced pathogen root infection (detected by real-time PCR). The same procedure applied in a commercial ﬁeld was less beneﬁcial in rescuing the plants’ growth and yield. Still, it reduced the cobs’ symptoms by 11% and resulted in nine-fold lower levels of the pathogen’s DNA in the stem tissue. Second, the T. asperellum puriﬁed 6-PP compound (30 µ g/seed) was used in seed coating and tested against the T. asperellum secretory metabolites’ crude (diluted to 50%). At the season’s end, these treatments improved plant biomass by 90–120% and cob weight by 60%. Moreover, the treatments signiﬁcantly ( p < 0.05) reduced the symptoms (up to 20%) and pathogen infection (94–98%). The current study’s results reveal the potential of 6-PP as a new fungicide against M. maydis . Such a treatment may protect maize plants from other soil diseases. design and analyzed using the same statistical method. Statistical processing and data evaluation were achieved using JMP software, version 15 (SAS Institute Inc., Cary, NC, USA). The results were evaluated using the one-way analysis of variance (ANOVA) followed by a Student’s t-test post hoc (without correction for multiple tests) at a signiﬁcance level of p < 0.05. Due to the difﬁculty in creating uniform plant infection, high variability in M. maydis ’s DNA levels and high standard error values are expected, making it challenging to obtain the alternative hypothesis.


Introduction
The world's corn (Zea mays L., maize) harvested area is the third largest after wheat and rice (FAOSTAT, 2020 Food and Agriculture Commodity Production data), reflecting its importance globally. Maize late-wilt disease (LWD) can cause acute damage to susceptible hybrids in infected areas under favorable conditions. Such a scenario prevails in Egypt [1,2], Israel [3], Portugal [4], Spain [5], India [6], and other less impacted regions [7,8]. LWD is typified by rapid dehydration of fodder and sweet maize, typically from the tasseling stage until just before maturity [9,10]. The disease's causal agent is the fungus Magnaporthiopsis maydis, which is recognized by two additional synonyms, Cephalosporium maydis and Harpophora maydis [11][12][13]. The fungus is a hemibiotroph, soilborne [14], and seed-borne [15], and can be spread as sclerotia, conidia, or hype on plant remains. The pathogen can survive for prolonged periods in the soil or by establishing inside alternative host plants, such as Lupinus termis L. (lupine) [16], Setaria viridis (green foxtail), Citrullus lanatus (watermelon), and Gossypium hirsutum L. (cotton) [17,18]. It may act as an endophyte in those secondary hosts [19] but also as an opportunist, causing severe disease in some environments and under host-associated conditions [20,21]. It was demonstrated that M. maydis is present in the host tissues of successfully chemically treated plants [52]. This may result in the development of the fungous's fungicide immunity. The most effective fungicide against the late-wilt agent is Azoxystrobin [52,53]. Still, the rapid expansion of this fungicide tolerance has become increasingly problematic. Furthermore, the antifungals' widespread use creates a major concern-their remaining toxicity and influence are endangering human health and the environment [54]. It was demonstrated that M. maydis is present in the host tissues of successfully chemically treated plants [52]. This may result in the development of the fungous's fungicide immunity. The most effective fungicide against the late-wilt agent is Azoxystrobin [52,53]. Still, the rapid expansion of this fungicide tolerance has become increasingly problematic. Furthermore, the antifungals' widespread use creates a major concern-their remaining toxicity and influence are endangering human health and the environment [54].
Several studies have already demonstrated microorganism-based LWD bio-control (most recently [1,34,[55][56][57][58]). A particularly studied method is based on plant growthpromoting rhizobacteria, which could also enhance plant health (summarized by [59]). An alternative approach is using Trichoderma species against the LWD pathogen. Members in this genus can develop endophytic mutualistic interactions with various plant species [60], while other species have been used as a bio-protective barrier against phytoparasitic fungi [61]. Still, many knowledge gaps exist regarding the effectiveness of Trichoderma spp. against M. maydis. Therefore, their full potential as a biopesticide is yet to be explored. To demonstrate this potential, it was shown [33] that adding Chlorella vulgaris (microalgae) extracts to each of the Trichoderma species T. koningii and T. virens was an efficient treatment against LWD, resulting in a 72% disease frequency reduction in the greenhouse and 2.5-fold higher grain yield in the field.
Over the past few years, we tested nine Trichoderma isolates versus M. maydis. Three of these isolates revealed control ability against the maize pathogen: Trichoderma asperelloides (T.203) and Trichoderma longibrachiatum (T.7407 from a marine sponge); and Trichoderma asperellum (strain P1), an endophytic symbiont isolated from the seeds of an LWD-sensitive maize cultivar [57,58]. Growth-room seedlings pathogenicity trials show that all three Trichoderma isolates promoted the inspected plants' growth indices, reduced LWD dehydration signs, and caused a significant reduction in M. maydis DNA inside the host tissues. Similar results were achieved in a full-season field trial (82 days) [55].
In follow-up work, we isolated a potent inhibitor chloroform extract (approx. 400 mg/L) from T. asperellum growth medium, capable of entirely preventing M. maydis growing [56]. Liquid chromatography-mass spectrometry (LC-MS) and gas chromatographymass spectrometry (GC-MS) purification steps identified the secreted active ingredient as 6-pentyl-α-pyrone (also called pyrone 6-pentyl-2H-pyran-2-one or 6-PP). The pure compound exhibited high antifungal efficiency versus the LWD pathogen. Based on reports in the literature, this active ingredient is identified as a key bioactive compound of various Trichoderma species [62].
This metabolite plays a pivotal role in the biocontrol capability of the producing species. It displays multiple actions against phytoparasitic fungi, such as restricting their spore germination, mycelium pigmentation, and hyphal growth [63]. 6-PP also reduces fusaric acid production by Fusarium moniliform and mycotoxin deoxynivalenol by Fusarium graminearum [64]. For commercial production, a large amount of 6-PP can be generated by T. asperellum by utilizing forced aeration on a solid-state fermentation system [62]. Because 6-PP is a food-grade volatile compound, it may also be applied post-harvest to shield crops in storage.
The current study aimed at developing a biological environmentally friendly control strategy against M. maydis LWD based directly on T. asperellum, its secreted metabolites' crude, or pure 6-PP. To this end, we conducted growth room trials in seedlings (up to 42 days) and full-season experiments in a net house or the field. Tracking M. maydis's DNA within the host tissues using quantitative real-time PCR (q-PCR) enables direct evaluation of the treatments' potential.

Rationale and Research Design
This three-year study examined the biocontrol potential of Trichoderma asperellum (P1 strain) and its secreted metabolites, specifically the 6-pentyl-α-pyrone (6-PP, Sigma-Aldrich, CAS number 27593-23-3, Buchs, Switzerland) purified active ingredient, against the maize late wilt pathogen, Magnaporthiopsis maydis. A test series was conducted to evaluate the potential of this new green biopesticide. These trials started by directly applying T. asperellum with the seeding in sprouts' pathogenicity evaluation (growth room conditions up to 42 days) and under field conditions for the entire growing season. The following step examined the T. asperellum secreted metabolites (growth medium crude) against the purified active ingredient, 6-PP, in a semi-field environment (net house) full-season experiment. While the growing room provides a controlled environment, the net house offers a better field-like environment while maintaining more uniform environmental conditions and better regulation of soil composition, inoculation load, and water management. Both trials enable adding non-infected plants as a negative control. Therefore, it is an important step designed to achieve a complete image. Field experiments, while providing a more actual commercial growth environment, are unstable regarding physical and climatic conditions, pathogen existence, and other factors (e.g., soil composition and microbiome), as well as the cross-influence between treatments.
The preliminary steps-inspecting T. asperellum biocontrol potential using the sprout pathogenicity assay (in a growing room up to 42 days from sowing, DAS) and the field trial-were conducted using the late-wilt-susceptible maize genotype Prelude (a sweet maize cultivar produced by SRS Snowy River Seeds, Australia, marketed by Green 2000 Ltd., Bitan Aharon, Israel). The T. asperellum secreted metabolites' examination in the net house was made using the sweet maize genotype Megaton cv. (from Limagrain, Saint-Beauzire, Puy-de-Dôme, France, supplied by Hazera Seeds Ltd.). These two cultivars were tested previously for their susceptibility to the late wilt pathogen [41,52] and identified as LWD-highly sensitive hybrids. A special effort was devoted to maintaining similar conditions in the growth room and mesh house experiments. Both trials were conducted based on the same soil conditions, inoculation method, and controls.

Fungal Species and Growth Conditions
All M. maydis isolates (Table 1) chosen for the current work were examined in our lab for their aggressiveness towards LWD-susceptible maize hybrids. They were found to have moderate pathogenicity levels (Ofir Degani, personal communication). The T. asperellum (P1 strain) endophyte was separated from the sweet maize Prelude cv. grains and identified as described before [58].  [55,58] The fungi were grown on solid potato dextrose agar (PDA; Difco Laboratories, Detroit, MI, USA) under dark conditions at an optimal temperature of 28 ± 1 • C. They were transferred to a new growth dish by extracting a 6-mm-diameter agar disk from the margins of the culture and sowing it to a new PDA Petri dish. Plates were labeled and incubated in the dark at 28 ± 1 • C for 4-6 days. For submerged cultures (in a liquid substrate), five fungal disks were sown in an Erlenmeyer bottle containing 150-mL potato dextrose broth (PDB; Difco Laboratories, Detroit, MI, USA). The bottles were plugged with a breathable stopper and incubated for six days, shaken at 150 rpm in the dark, at 28 ± 1 • C. The growth medium metabolites were extracted by filtering the liquid PDB growth medium using a Buchner funnel and Whatman filter no. 3.

Inoculation Method
Two-liter pots were filled with commercial field peat soil from a location with a long record of LWD infection (Amir field, Mehogi-1 plot, coordinates: 33 • 09 59 N 35 • 36 5 E) [52,53]. Control was a similar peat soil from a nearby farm (Gadash experimental, coordinates: 33 • 10 48.6 N 35 • 35 11.6 E) with no record of LWD. If such an infestation occurred, it was supposed to be minor. The soil was blended with 30% Perlite No. 4 (for aerating). Complementary inoculation was conducted in two steps to guarantee a high and uniform infection load: (1) pre-sowing soil infection with sterilized infected wheat grains; and (2) adding M. maydis colony agar disks with the seeding. The sterilized infected wheat grains were prepared as previously described [41]. Briefly, the wheat seeds were drenched in tap water overnight, filtered, and dried on paper towels in a fume hood. The seeds were sterilized in an autoclave for 30 min at 120 • C, and 150 g was incubated with 10 M. maydis mycelium agar disks of M. maydis (see Section 2.2) in sterilized plastic boxes (0.5 l volume) for 2-3 weeks at 28 ± 1 • C in the dark. The seeds were mixed every two days by shaking to ensure equal inoculation. The soil was infected by adding and mixing 12 g of sterilized and infected wheat grains to the top 10 cm of the ground in 2 L pots. The inoculated soil was kept moist until the maize was sown for a week. Additionally, three colony M. maydis colony agar disks were added to each seed with the sowing.

Trichoderma asperellum Biocontrol-Based Treatment
The biocontrol agent, T. asperellum, was grown in a liquid PDB (see Section 2.2) for six days, filtered, and chopped into small hyphae fragments, and 1 g (wet weight) of mycelia was added to each seed with the sowing.

Growth Room Trial Conditions
The experiment was conducted in 2019 in five biological repetitions and included two controls-infected unprotected plants and healthy plants (without any treatment). Two-liter pots were filled with field peat soil mixed with 30% Perlite, as in Section 2.3.1. Five Prelude cv. maize seeds were seeded in each pot to a depth of 4 cm. The plants were developed in a growing room for 42 days (fifth leaf appearance, V5 phenological stage) in an artificial light regime of 16 and 8 h of darkness, at 28 ± 3 • C with 45-50% humidity. Each pot was irrigated routinely with 100 mL of tap water every two days.

Overall Description of the Field Experiments
This study evaluated the T. asperellum biocontrol capability against M. maydis over a full growing season in a commercial field conditions simulation. The experiment was carried out on a trial farm (Gadash, Hula Valley, Upper Galilee, north Israel) throughout the summer and fall of 2020. The meteorological records through the growth period are listed in Table 2. The 2020 trial was conducted in autumn for pragmatic reasons (principally the field availability). Thus, due to the late season, the average meteorological parameters quantified during the experimental duration (relatively low temperature, radiation and evaporation, and high precipitation) were not optimal for the LWD burst. The experiment field area has a history of mild LWD infestation. We infected the experimental plots (apart from the control plots) to attain a more acute disease. Therefore, the negative check plots were without pathogen enrichment. The Prelude cv. seeds were pre-treated (according to the regulations) with thiram, captan, carboxin, metalaxyl-M (Rogers/Syngenta Seeds, Boise, ID, the USA, provided by CTS, Tel Aviv, Israel). The Agronomy 2022, 12, 2339 6 of 20 experiment was performed with five random blocks per treatment/control. Each block comprised five garden beds (repeats), which were 9 m long, 6 m wide, and had two rows with row spacing of 96 cm.

Sowing Procedure and Irrigation
The plots were seeded to a 4 cm depth with 7 plants/meter and sprouted the next day with a frontal irrigation system. Irrigation of 0.6 l h −1 was conducted by drip irrigation line (20 mm) for each row (Dripnet PC1613 F, Netafim USA, Fresno, CA) and was controlled using a computerized system. The watering supply was 3000 L/0.1 ha per day. The watering was conducted every two days, and the overall water supplied throughout the season was ca. 400 mm. All the plants received insecticides and fertilization at the recommended dosages (Consultation Service, SAHAM, Israel Ministry of Agriculture).

Complementary Inoculation Method
The plants' infection methodology and growth were according to [17,53]. The inoculation procedure includes further complementary phases to equalize and enhance the soil's pathogen infection. To this end, the trial plots (excluding the negative check) were inoculated by adding 3 M. maydis-infected wheat grains to each corn seed with the seeding. These grains were prepared as detailed in Section 2.3.1 with a blend of M. maydis isolates (20 disks/150 g seeds), Hm2, Hm29, and Hm30 (see Table 1).

Trichoderma-Based Biocontrol Treatments
The bio-shield treatment was conducted by adding 3 sterilized wheat grains enriched with T. asperellum to each maize seed with the seeding. The wheat grain enrichment was conducted in a similar fashion to the pathogen enrichment described in Section 2.3.1.

Data Collection
Pollination occurred on 49 DAS (29 October 2020). During the season (up to the harvest), most plants had a healthy, vital appearance, typical growth, and crops (detailed in the Results) at the season's ending. Yet, close examination of the lower stalk (first internode above the ground) and cobs' spathes (the large bracts surrounding the cobs) revealed disease symptoms. Thus, the healthy (non-symptomatic) plant percentages were calculated and presented. Growth indexes (shoot height and wet weight, and the number of leaves) were determined in 5 plants (one repeat) selected randomly from the middle of every one of the experiment's garden beds. Each experimental group (controls and biological intervention) included eight repetitions. Yield evaluation was conducted on all the plants in 4-7 rows (repeats) for the biological treatment and the controls. The molecular tracking (M. maydis DNA diagnosis) was performed on three arbitrarily selected representative plants in those rows.

Effect of Trichoderma Asperellum-Secreted Metabolites in Solid Media Cultures
The impact of T. asperellum-secreted metabolites on M. maydis, M. phaseolina, and F. verticillioides growth was assessed in solid growth media (PDA) as previously described [56]. To prepare the T. asperellum's secreted metabolites, five mycelial discs (6 mm) were removed from the edges of 2-4-day-old colonies and incubated for six days in 150 mL PDB in the dark at 150 rpm at a temperature of 28 ± 1 • C. The growth medium was separated by Buchner funnel filtration through Whatman filter no. 3. The medium pH was adjusted to 5.1 ± 0.2 with NaOH and filtered again for sterilization using biofilter bottles (0.22-micron filter, BIOFIL 500 mL vacuum bottle filter, Indore, India). The Trichoderma cultures' filtrate described above was used to prepare the PDA plates (instead of DDW). PDA powder was added to the filtrate according to the manufacturer's instructions. After autoclave sterilization, the mixture was poured into Petri plates, which were seeded with a 6-mm-diameter M. maydis mycelial disc (from the margin of a fungus colony, see Section 2.2). The colonies were allowed to grow for six days and photographed.
2.6. 6-pentyl-α-pyrone Evaluation in a Net House 2.6.1. Application of the Purified T. asperellum Antifungal Compound(s) Using Seed Dressing The T. asperellum isolate activity products were tested in a net house in pots during a full growth period. The experiment included six replicates (pots). The Megaton cv. corn (sweet, LWD super-susceptible) [41] was used in the experiments. Seed coating was performed according to a standard commercial procedure by blending the seeds with the compound (0.002 mL preparation/seed) until it was ultimately absorbed in the seeds. The inspected ingredient (30 or 60 µg 6-PP, and 100% or 50% T. asperellum crude, in 2 mL doubledistilled water, DDW) was pipetted into a one-liter round-bottom glass flask containing 1000 seeds. The flask was rotated for ca. 10 min until the inspected ingredient was fully absorbed in the seeds. The seeds were taken out and dried in a fume hood on sterile paper towels for several hours. These seeds were kept in paper bags at 4 • C until use. The control treatment was conducted with similar seeds that underwent the addition of sterile water instead of the inspected ingredient.

Net House Full-Growth Season Pot Experiments Treatments and Sampling Program
The experiment was conducted in the R&D North Israel Experimental Farm located in the Hula Valley, Upper Galilee, northern Israel (33 • 09 08.2" N 35 • 37 21.6" E) during a full growing season. Each treatment consists of ten independent replicates (pots). The sprouts were thinned to one plant per pot towards the end of the sprouting growth stage (40 DAS). Inoculation with the M. maydis was carried out as described for the growth room experiment, with the following changes: in the first inoculation step, 40 g of infected sterilized wheat grains was added to the upper 20 cm of the soil of each pot. Control was peat soil from the experimental farm, having no record of LWD. If such an infestation existed, it was assumed to be very low. The evaluation of emergence (aboveground surface peek) percentages was performed 6 DAS. The male flowering was documented at 53 DAS. The growth parameters were evaluated at 40 DAS and the experiment end (96 DAS). Yield assessment and wilt determination were performed at harvest. Wilt valuation for each plant was based on 4 classifications-dead, diseased, symptoms, and healthy-as was demonstrated in [55], and the dry plants' percentages were calculated and presented. On both sampling days, a qPCR detection of the fungal DNA in the plants' roots or aboveground first internode was performed.

Net House Growth Conditions
The experiment's local peat soil was similar to the growth room trial soil. It had no recognized record of LWD infestation (if it existed, it was assumed to be very low). To aerate the ground, the soil was blended with coarse perlite (No. 4) at a ratio of 2:1 in 10 L pots. The pots were positioned on concrete blocks to prevent the roots from contacting the ground and to better isolate the treatments. Each pot was sown with 5 seeds previously coated with thiram, captan, carboxin and metalaxyl-M. This seed treatment is standard general pesticide care. Seed samples were tested for vitality before seeding. Computerized irrigation was conducted with drip lines and was carried out from the seeding using 2 L per pot/day. According to the plants' needs, adjustments were made in the water regime and pesticide treatments to prevent other diseases from influencing the plants during their growth (according to the Ministry of Agriculture's recommended growing protocol). During the maize growing season, the average net house temperature was 24.7 • C, with a maximum of 43.4 • C and a minimum of 12.3 • C. The average humidity was 93.6%, with a maximum of 100% and a minimum of 42.2%.

Molecular Analysis
Quantifying M. maydis DNA in the host plant tissues was performed using the qPCRbased technique. The plants' parts were washed vigorously with running tap water, then twice with sterile DDW and sliced into ca. 2 cm sections. The total weight of each repeat was adjusted to 0.7 g. The pathogen DNA was isolated and extracted according to a previously published protocol [66] with slight modifications [65]. Briefly, 0.7 g of plant tissue was ground with 4 mL of cetyltrimethyl ammonium bromide (CTAB). A total of 1.2 mL was transferred to Eppendorf test tubes and heated in a bath at 65 • C for 20 min. The test tubes were concentrated at 13,000 rpm at room temperature (24 • C) for 5 min, and the upper phase (700 µL) was transferred to new test tubes with the same volume of chloroform/isoamyl-alcohol (24: 1). The last cleaning stage was repeated twice. The upper phase (300 µL) was transferred to a new Eppendorf test tube with 200 µL of cold isopropanol, and the test tubes were maintained at 20 • C for 20-60 min. The test tubes were then concentrated at 13,000 rpm at 4 • C for 20 min, and the liquid was poured into a waste vessel. The DNA precipitate was resuspended and washed with 0.5 mL of ethanol. After a further concentration and ethanol wash cycle, the DNA precipitate in the test tubes was allowed to dry in a sterile hood overnight. Finally, the DNA was resuspended in 100 µL of ultra-pure water and kept at −20 • C until used for qPCR. DNA extraction from PDA colonies (positive control) was performed using a Plant/Fungi DNA Isolation Kit (Norgen, Biotek Corporation, Thorold, Ontario, Canada) according to the manufacturer's instructions.
The molecular detection technique is based on a standard qPCR procedure to measure mRNA (converted to cDNA) levels [67]. It was adjusted here to detect M. maydis DNA using species-specific primers [68,69]. The A200a primers were applied for qPCR ( Table 3). The housekeeping cytochrome C oxidase (COX) gene, encoding the cellular mitochondria' last enzyme in the respiratory electron transport chain, served to normalize the relative M. maydis DNA amount in the samples examined [70]. For the COX gene COX F/R primer set, see Table 3. The relative DNA abundance was calculated using the ∆Ct model [67,71]. All amplifications were carried out in four repetitions, and the same efficiency was assumed. All qPCR reactions were measured using the ABI PRISM 7900 HT Sequence Detection System (Applied Biosystems, Foster City, CA, USA) and 384-well plates. The reaction volume was 5 µL per well. All wells contained 0.25 µL of each primer (Forward/Backward at 10 µM concentration), 2.5 µL iTaq™ Universal SYBR Green Supermix solution (Bio-Rad Laboratories Ltd., Hercules, California, USA), and 2 µL of DNA sample. Reaction conditions were 95 • C for 60 s (pre-cycle activation phase), then 40 cycles of 95 • C for 15 s (denaturation), 60 • C for 30 s (annealing and extension), and, finally, the formation of a melting curve.

Statistical Analysis
The growth room and net house experiment pots and the field plots were scattered in a fully randomized design and analyzed using the same statistical method. Statistical processing and data evaluation were achieved using JMP software, version 15 (SAS Institute Inc., Cary, NC, USA). The results were evaluated using the one-way analysis of variance (ANOVA) followed by a Student's t-test post hoc (without correction for multiple tests) at a significance level of p < 0.05. Due to the difficulty in creating uniform plant infection, high variability in M. maydis's DNA levels and high standard error values are expected, making it challenging to obtain the alternative hypothesis.

Results
The current study aimed at evaluating the Trichoderma asperellum secreted metabolite, 6pentyl-α-pyrone (6-PP), against the maize late wilt (LWD) pathogen, Magnaporthiopsis maydis. To achieve this goal, a series of trials were conducted, starting by inspecting the addition of T. asperellum directly to seeds with the sowing (sprouts in a growth room-controlled conditions and complete season field experiment). These trials were followed by a targeted evaluation of the T. asperellum recently purified and identified active secreted metabolite, 6-PP (net house experiment over an entire growing season). The powerful antifungal compound was applied in seed coating and tested against the T. asperellum growth medium crude or by adding the fungus itself with the seeding. The results indicate a potential new control treatment to protect susceptible maize plants from LWD, as detailed below.

Trichoderma Asperellum Pathogenicity Evaluation in Growth Room Sprouts
Young T. asperellum hyphae disks were cut from the margins of culture colonies, grown on rich medium plates, and added directly to the seeds with the sowing. Inspecting this procedure's impact on seedling development (up to 42 days) and M. maydis establishment in their roots revealed a significant growth promotion and LWD recovery (Table 4). While the T. asperellum did not affect the aboveground emergence (at day 8), at the end of the sprouting phase (day 42, the fifth leaf stage), all growth indexes improved drastically (p < 0.05) compared to the infected, untreated plants. This improvement ranges from 1.8-fold in plant height to 2.7 and 3.3-fold in shoot and root fresh weight. These changes were accompanied by a two-fold reduction in the M. maydis DNA in the sprouts' roots.

Trichoderma Asperellum Pathogenicity Evaluation in the Field
The follow-up full-season trial was conducted in an experimental field known to have a mild infection with late wilt. Since the growing season's climatic conditions were not optimal for this disease development (relatively rainy autumn season), we infected the field soil with the seeding. Still, during the growth period up to the harvest, the plants' phenological development and growth indexes were normal, and the disease symptoms were minor. These parameters did not vary statistically between the treatments (Table 5).
Only a close examination of the lower stem and cob spathes (the large bracts surrounding the cobs) revealed the disease dehydration symptoms and the impact of the T. asperellum treatment (Figure 2A). At the season's end (day 82, 33 days after fertilization), the improvement in these two parameters (lower stem and cob symptoms) was 1.6and 1.3-fold, respectively, in the biological treatment compared to the non-treated control (without statistical significance). In line with these results is the M. maydis's DNA in the plants' first aboveground internode ( Figure 2B, qPCR results). In the T. asperellum treatment, the pathogen DNA dropped by five-fold compared to the control. This being said, the pathogen DNA levels in all treatments were relatively low and near the technique's lower threshold (the qPCR method can detect variation in proportionate M. maydis specific DNA between 10 to 10 −5 [7]).

6-pentyl-α-pyrone Evaluation in a Net House
So far, we have evaluated the addition of T. asperellum directly to the seeds wit ing. The following steps were taken to inspect its secreted metabolites' bio-protectiv ity under late wilt stress. The potential of this Trichoderma species growth medium and its active ingredient 6-PP to restrict the LWD pathogen, M. maydis, in vitro wa viously demonstrated [56]. It now appears that T. asperellum growth medium ex specificity activity against M. maydis and cannot repress two additional members maize post-flowering stalk rot complex [73], Fusarium verticillioides and Macroph phaseolina (Figure 3).

6-pentyl-α-pyrone Evaluation in a Net House
So far, we have evaluated the addition of T. asperellum directly to the seeds with sowing. The following steps were taken to inspect its secreted metabolites' bio-protective ability under late wilt stress. The potential of this Trichoderma species growth medium crude and its active ingredient 6-PP to restrict the LWD pathogen, M. maydis, in vitro was previously demonstrated [56]. It now appears that T. asperellum growth medium exhibits specificity activity against M. maydis and cannot repress two additional members of the maize post-flowering stalk rot complex [73], Fusarium verticillioides and Macrophomina phaseolina (Figure 3).
ing. The following steps were taken to inspect its secreted metabolites' bio-protective ability under late wilt stress. The potential of this Trichoderma species growth medium crude and its active ingredient 6-PP to restrict the LWD pathogen, M. maydis, in vitro was previously demonstrated [56]. It now appears that T. asperellum growth medium exhibits specificity activity against M. maydis and cannot repress two additional members of the maize post-flowering stalk rot complex [73], Fusarium verticillioides and Macrophomina phaseolina (Figure 3).  Until now, the T. asperellum secreted ingredients had never been tested as a shield against the LWD agent in plants. To accomplish this, we conducted a net house wholeseason experiment. We tested the purified 6-PP metabolite against the growth medium crude and the direct addition of the Trichoderma to the seeds (Figure 4). The disease progression in this experiment was evident, and already at the end of the sprouting phase (day 40), significant growth reduction (p < 0.05) was observed in the infected, non-protected plants group (Table 6).  1 The experiment was performed in late wilt-susceptible Megaton cv. The aboveground first emergence (peak) was recorded at 6 DAS. All other parameters were measured at 40 DAS. Non-infected are plots without inoculation. Infected are plots with M. maydis inoculation. The additional treatments are infected plots with T. asperellum (P1) secreted metabolites 6-pentyl-α-pyrone (6-PP), secreted crude (Ex. 50%-diluted to 50% and Ex. 100% undiluted), or the addition of three T. asperellum colony agar disks directly to the seeds with sowing (P1 control). Values represent an average of 10 replications ± standard error per pot. Statistically significant differences (p < 0.05) between treatments are indicated by different letters (A-C).
season experiment. We tested the purified 6-PP metabolite against the growth m crude and the direct addition of the Trichoderma to the seeds (Figure 4). The disea gression in this experiment was evident, and already at the end of the sproutin (day 40), significant growth reduction (p < 0.05) was observed in the infected, n tected plants group (Table 6). At the V5 (five leaves) stage, the unprotected plants suffer a 37% abovegroun decrease (6 DAS), a 64% lower survival rate, and 38-39% growth repression (40 D ble 6). All the bio-treatments led to some recovery and improvement in these para especially the 6-PP (30 µg), the crude (diluted to 50%), and the T. asperellum add the seeds. High dosages either of the clean 6-PP metabolite or the growth medium were apparently less effective, perhaps due to overdose, which can lead to phytot At the V5 (five leaves) stage, the unprotected plants suffer a 37% aboveground peak decrease (6 DAS), a 64% lower survival rate, and 38-39% growth repression (40 DAS, Table 6). All the bio-treatments led to some recovery and improvement in these parameters, especially the 6-PP (30 µg), the crude (diluted to 50%), and the T. asperellum addition to the seeds. High dosages either of the clean 6-PP metabolite or the growth medium crude were apparently less effective, perhaps due to overdose, which can lead to phytotoxicity.
In a final evaluation made at the season's end (day 96, 43 days post fertilization), the tendencies that started to be revealed on day 40 were enhanced and became significant (p < 0.05, Figure 5). Here, the bioprotective 30 µg 6-PP and 50% crude extract had similar (and even higher) results compared to the healthy plants' control, while the non-treated group had up to 123% and 64% lower shoot and cob weights, respectively. Parallel plant health increases accompanied these dramatic growth changes (Figure 6).
In a final evaluation made at the season's end (day 96, 43 days post fertilization) tendencies that started to be revealed on day 40 were enhanced and became significa < 0.05, Figure 5). Here, the bioprotective 30 µg 6-PP and 50% crude extract had sim (and even higher) results compared to the healthy plants' control, while the non-tre group had up to 123% and 64% lower shoot and cob weights, respectively. Parallel p health increases accompanied these dramatic growth changes ( Figure 6). Figure 5. The Trichoderma asperellum secreted metabolite, 6-pentyl-α-pyrone, impacts maize d opment under late wilt disease stress. Net house study, day 96 data. The purified active ingred 6-PP was tested as seed coating at 30 and 60 µg/seed and compared to the T. asperellum (P1) d addition to seeds or its crude secreted metabolites applied at 50% or 100% concentrations (Ex. and Ex. 100%). The standard error of the mean of 7-10 replicates (plants) is shown. Highlig Figure 5. The Trichoderma asperellum secreted metabolite, 6-pentyl-α-pyrone, impacts maize development under late wilt disease stress. Net house study, day 96 data. The purified active ingredient 6-PP was tested as seed coating at 30 and 60 µg/seed and compared to the T. asperellum (P1) direct addition to seeds or its crude secreted metabolites applied at 50% or 100% concentrations (Ex. 50% and Ex. 100%). The standard error of the mean of 7-10 replicates (plants) is shown. Highlighted values (in yellow) are important differences from the infected, untreated control. Statistically significant differences (p < 0.05) between treatments are specified by different letters (A-C).  The dehydration levels of the plants were attributed to variations in the M. mayd DNA load tracked by the molecular qPCR method. As revealed by the highly sensitiv qPCR method, some very low levels of M. maydis DNA (near the method's lower sensiti ity threshold) could be identified in the control treatment. However, such low levels we The dehydration levels of the plants were attributed to variations in the M. maydis DNA load tracked by the molecular qPCR method. As revealed by the highly sensitive qPCR method, some very low levels of M. maydis DNA (near the method's lower sensitivity threshold) could be identified in the control treatment. However, such low levels were unable to evoke disease symptoms. High pathogen DNA was measured (at days 40 and 96) in the infected control plants group and the plants enriched with T. asperellum on the sowing day (Figure 7). This last result is unexpected since the T. asperellum addition to the seeds provides some protection against LWD growth reduction at the sprouting phase (40 DAS, Table 6). In contrast, the other treatments' parameters fit well with the growth and health results. At harvest (day 96), the crude extract (especially the 50% dilution) had the best influence in repressing the pathogen (43-fold). Here, too, the 6-PP diluted (30 µg) seed coating was more efficient (17-fold) than the 6-PP 60 µg treatment (7-fold M. maydis decrease).

Discussion
Natural microorganisms' communities inhabiting plants include non-pathogenic members that can have shielding effects against pathogens. This work examined a new biological control agent candidate against the maize late wilt disease (LWD) pathogen, Magnaporthiopsis maydis. This pathogen control is challenging due to long-term soil survival, and resistant cultivars are constantly being threatened by aggressive pathogen variants [24].
In recent years, biopesticides have achieved significant consideration as an alternative to environmentally problematic chemical pesticides [54]. Understanding the roles of endophytic community assemblages and their impact on plant health would be a major contribution to sustainable integrated agriculture. Trichoderma species are used widely in agriculture to promote plant growth and control pathogens attacking crops, serving as valuable tools for sustainable food production [74]. Still, the action mechanism of this bio-

Discussion
Natural microorganisms' communities inhabiting plants include non-pathogenic members that can have shielding effects against pathogens. This work examined a new biological control agent candidate against the maize late wilt disease (LWD) pathogen, Magnaporthiopsis maydis. This pathogen control is challenging due to long-term soil survival, and resistant cultivars are constantly being threatened by aggressive pathogen variants [24].
In recent years, biopesticides have achieved significant consideration as an alternative to environmentally problematic chemical pesticides [54]. Understanding the roles of endophytic community assemblages and their impact on plant health would be a major contribution to sustainable integrated agriculture. Trichoderma species are used widely in agriculture to promote plant growth and control pathogens attacking crops, serving as valuable tools for sustainable food production [74]. Still, the action mechanism of this bio-protective species is poorly understood.
The maize-biofriendly species, T. asperellum, is an endophyte isolated in Israel from LWD-susceptible maize genotype seeds [58]. Here, this Trichoderma species showed protective ability against Magnaporthiopsis maydis in growth room sprouts and mature plants in the field. However, it is inferred from the results that this protection was affected most at the sprouting phase and that under some field conditions, it may fail. Obvious differences exist between the growth room and the field conditions that may explain the results presented here. As a start, the field's unstable conditions may have a significant impact on the results. In our study, the field conditions were not optimal for LWD development, leading to weak dehydration symptoms appearing at the harvest. Indeed, this growing season was rainier and colder, and these circumstances were possibly the reason for lowered yields and a minor LWD outbreak. To support this, early (spring) sowing of corn in Egypt [75] and late summer planting in India [27] reduced LWD. It is possible that the full potential of the T. asperellum-based biological control was not manifested under these conditions.
Inspecting similar research results published recently by Estévez-Geffriaud (2021) [76] provides evidence that T. asperellum (strain T34 applied as seed treatment) positively affected maize productivity and health under late wilt disease stress. Regardless of the water regime, T34 improved kernel P and C content, kernel dry weight, and number. Under drought, this Trichoderma treatment improved leaf relative water content, water use efficiency, PSII maximum efficiency, and photosynthesis. Moreover, T34 reduced LWD incidence by up to 13% and enhanced the yield per plant in LWD moderate tolerant and resistant maize genotypes. Still, no yield improvement was measured in susceptible lines [76]. It may be inferred from Estévez-Geffriaud and other studies (for example, [33]) that the Trichoderma species, the specific strain, the plant's LWD susceptibility degree, and environmental conditions could all affect the success of the biological treatment.
It should also be considered that T. asperellum protection is mainly affected during the sprouting stage, which is regarded as the pathogen entry and establishment phase [21,23]. Later in the season, this initial protection may lose effectiveness if the pathogen finds a way to establish itself in the plant's vascular system and disrupt its development. If such a scenario is the case, integrated biological and chemical protection may assist in overcoming the pathogen's establishment and provide us with the benefit of low fungicides usage with a live protective barrier [54].
Many other factors could affect this outcome (the differences between the growth room and field assays). For example, the time lapse between pathogen and antagonist inoculation can be crucial. The preventive and curative abilities of T. asperellum UDEAGIEM-H01 versus Fusarium oxysporum were evaluated in stevia plants (Stevia rebaudiana) under field conditions. The occurrence of Fusarium wilt was 10% in the protective treatment when the antagonist was inoculated six days prior to the pathogen, whereas 70% of disease occurrence was recorded in plants treated with the antagonist six days after the infection of seedlings with the pathogen [77]. Thus, controlled conditions experiments, as demonstrated here, are difficult to replicate on a field scale where pathogens are already present [74]. Further studies are needed to optimize the maize growing time at which T. asperellum could present the best LWD biocontrol abilities in the field.
One of the advances in using Trichoderma-based protection against LWD is that these species likely maintain a symbiotic lifestyle inside the host plant organs that may provide a season-long defense. Moreover, the Trichoderma species can trigger the maize disease resistance response systemically through the biosynthetic induction of brassinolide, jasmonic acid, salicylic acid, defense enzymes, and reactive oxygen species [78]. T. asperellum substantially impeded F. verticillioides growth in plate competition assay [79] and showed a biocontrol efficacy of 50% against Fusarium graminearum in the field [80]. These examples suggest that the field control application of T. asperellum may offer broad pathogen protection.
Yet, it should be considered that in severe cases, Trichoderma protection alone is insufficient to restrain the disease. When the pressure of infection is high, systemic fungicides must be applied. Trichoderma-based intervention should be part of an integrated management strategy, considering that some Trichoderma spp. are inefficient in combination with fungicides [54]. Compared to chemical pesticides, bio-based disease control does not generate resistance in pathogens, does not contaminate the environment, prevents the proliferation of secondary plagues, is compatible with organic production, and meets the requirements of profitable markets concerning maximum limits of chemical residues on fruits and vegetables. This work and others [55,56] suggest that T. asperellum bio-treatment against M. maydis is worth continuous future studies to maximize its potential. The findings presented in the current work imply that a major part of this antifungal activity is related to T. asperellum secreted metabolites in general and the 6-pentyl-α-pyrone (6-PP) active ingredient in particular.
Fungi are incredible producers of a huge range of xenobiotics, including some wellknown compounds that inhibit the growth of other fungi. Secondary metabolites are pivotal defensive microorganism-pathogen interactions during host invasion and establishment, as well as the plant microbiome defense response. These include antifungals, mycotoxins, lactams/lactones, and other extracellular components. Such a compound is 6-PP [81]. This metabolite was recognized as one of the major bioactive compounds of various Trichoderma species [62], with a clear correlation between 6-PP biosynthesis and biocontrol capability. 6-PP inhibits spore germination, mycelial growth, and pigmentation of tested phytopathogenic fungi [63]. 6-PP also decreases the production of the mycotoxin deoxynivalenol (DON) by F. graminearum and fusaric acid by F. moniliforme [64]. Our study results support the importance of this molecule and are opening up a new research direction that could lead to a new LWD bio-pesticide product.
Significant knowledge gaps still exist regarding the action mechanism of 6-PP. The compound is oxygenated and thus was proposed to be part of the oxylipin family [82,83]. This suggests that regardless of their source, similar oxylipin-like structure compounds may provoke strong chemotropic reactions in Trichoderma-and possibly additional fungal species. Thus they may establish an interspecies signaling mechanism. In mycoparasitism, it was implied [84] that the plant oxylipin recruits the mycoparasite towards roots affected by fungal phytopathogens-or already as a preventive measure. At the same time, the fungal oxylipin 6-PP induces morphological adjustments in Trichoderma needed to launch a localized mycoparasitic attack.
The 6-PP mass release at this stage would not only have a repressive antifungal action. It might also act as a highly secreted signal in the regions where the colony contacts the prey fungus to promote hyphal redirection and hyphal growth as part of the mycoparasitism response. Other secondary metabolites produced by T. asperellum are likely involved in these events to refine and support the chemoattraction process and the mycoparasitism attack [84].

Conclusions
This research targeted the maize late wilt disease (LWD) agent, Magnaporthiopsis maydis, using the biocontrol Trichoderma asperellum and its secreted metabolites. Growth room sprouts assay and field trials that were less effective demonstrated that adding this Trichoderma species directly to seeds can provide some protection against the disease. The bio-friendly Trichoderma control is most likely attributed to this organism's secreted metabolites since applying its growth medium extract in the seed coating provided the plants with significant protection. One of these ingredients is 6-pentyl-α-pyrone (6-PP), a potent antifungal compound tested here for the first time against M. maydis in infected plants. The success of the 6-PP seed coating was expressed not only in a drastic reduction in the pathogen's lower stalk infection but also in the nearly full recovery of the plant growth indices. A follow-up study should implement the 6-PP seed coating in commercial field simulations. Moreover, it would be most interesting and important to identify other active ingredients in the T. asperellum and other beneficial Trichoderma spp. (such as T. longibrachiatum) growing medium extract.

Data Availability Statement:
The datasets generated during and/or analyzed during the current study are available from the corresponding author upon reasonable request.