A Green Solution to Maize Late Wilt Disease

Introduction
Maize is one of the world’s leading crops for food, feed, and fuel and as a raw mate-rial for different industrial products [1]. Late wilt disease (LWD) has been reported in 10 countries (Figure 1) andis considered a major concern in highly infected countries such as Egypt [2], Israel. Economic losses due to LWD were up to 50-100% [3-6], and disease inci-dencescouldreach 100%.

Figure 1. World distribution map for Magnaporthiopsismaydis. Disease severity is appraised according to the literature reports and is based on three categories: severe (4, Egypt and Israel); moderate (3, India, Spain, and Portugal); minor (2, Hungary and Nepal); and not certain/unconfirmed reports (1, Italy, Romania, and Kenya)(adapted from [7]).

The late wilt causal agent, M. maydis, is a seed-borne and soil-borne vascular wilt fungal pathogen that penetrates the host roots and colonizes the xylem tissue [8,9]. Former scientific names are Cephalosporium maydis (Samra, Sabet,& Hing, 1963) [10] and Har-pophora maydis (Samra, Sabet,& Hing, 1963; Gams, 2000) [11].

The pathogen spreads as sclerotia, spores, or hyphae on the plants’ residues[9]. It can survive in the ground for lengthy periods or by thriving inside diverse host plants, such as lupine (Lupinus termis L.) [12], cotton[13,14], watermelon, and green foxtail(Setaria viridis)[13,15].
The disease mode in LWD-sensitive maize cultivars is well detailed in the scientific literature (Figure 2). A parallel asymptomatic infection mode, with some delay, occurs in resistant cultivars and can result in infected seeds that enhance the pathogen spread [4,5].

Figure 2. Disease cycle of maize late wilt caused by Magnaporthiopsismaydis(adapted from [7]).

Over the past 60 years, a vast effort dedicated to late wilt disease control was made [16]. The inspected control methods produced different degrees of success and include agritechnologicaloptions (flood fallowing and balanced soil fertility)[17,18], biofriendly approaches [19], physical (solar heating) [20], allelochemical [21], and chemical pesticide [22-24] practices.Recently, the tillage system’s impact, the cover crop,and the crop rotation have been shown to serve as bioprotective factors against M. maydis[25,26].A targeted re-search effort has led to a practical, efficient, and economic Azoxystrobin-based control protocol [4,24,27,28]. Notwithstanding this recent achievement, the intensive chemical in-tervention has several drawbacks. It may lead tofungicide resistanceIn the short term. It may also result in long-range environmental, human, and animal risks.

Currently, the most eco-friendly, cost-effective, and efficient method to restrict M. maydis is by using highly resistant maize varieties [29,30]. Yet, the discovery of M. maydis highly aggressive isolates [31-33] is a constant problem. These fungal strains may threaten resistant maize cultivars, especially when growing resistance cultivars in the same loca-tion for extended periods[24,34]. This alarming situation pushes researchers to continue seeking new methods to control LWD.
2. Maize Late Wilt Biological Control

Over the past years,many studies have beendirected towards LWD biological control [20,31,47-49].These methods include operating and strengthening beneficial microorgan-ism communities in the soil (for example, by compost addition [35]) or direct intervention using antagonistic bacteria and fungi or their secreted metabolites.
Indeed,late wilt disease can be biologically controlled using Trichodermaspp. To this end, we examined  nine marine [36]and soil isolates of Trichoderma spp., known for their high mycoparasitic potential. The study revealed that Trichoderma longibrachiatum (T7407) and Trichoderma asperelloides (T203) isolates have solid antagonistic activity against the Israeli M. maydis strain. These eco-friendly agents were tested by us in a series of experiments in the laboratory (Figure 3) and in a growth room until their final examination under field conditions throughout an entire growing season [37]. The T. longibrachiatum (T7407) green treatment significantly improved growth and yield indices to healthy plants’ levels, re-duced pathogen DNA in the plants’ tissues by 98%, and prevented disease symptoms (Figure 4).

Figure 3. In vitro estimation of Trichodermaasperelloides (T203)-secreted metabolites-based biolog-ical control against Magnaporthiopsismaydis(adapted from [37]). (A) T203-submerged cultures were grown with shaking (150 rpm) to isolate secreted metabolites. (B)Static shallow mediaculturesof M. maydis on rich liquid medium containing T203-secreted metabolites filtrate. Control is medium M. maydis cultures maintained under the same conditions. (C) Effect of growth media of T203 isolate on corn seed germination.The seeds were germinated in Petri dishes soaked in 4 mL of medi-um(control) or medium+ secretion products (growth medium filtrate six days after T203 growth). All images are displayed after 5–6 days of incubation at 28±1°C in the dark.

Figure 4. Trichoderma longibrachiatum (T7407) biological control against Magnaporthiopsismaydisin the lab and the field (adapted from [37,38]). (A) Plate mycoparasitism assay to identify interactions between Magnaporthiopsis maydis and T7407 in a solid, rich medium. The two fungi were placed opposite each other, T7407 on the left and M. maydis on the right. Photos were taken after 3 and 10 days of growth. (B) Field inoculation of 20-day-old seedlings byan M. maydis-infected toothpick. The toothpicks were used for stabbing each plant at the near-surface portion of the stem. (C) The lower stem (first aboveground internode) disease symptoms. (D) Thecobs’spathes disease symptoms. (E) The experiment’s plots. Representative images of the field plants were taken 82 days after sowing. Controls are unprotected diseased plants.

TheTrichoderma species can secrete soluble metabolites that inhibit or kill the maize pathogen(Figure 3B)[37]. Such a metabolite was recently isolated by us and identified as 6-pentyl-α-pyrone (6-PP, Figure 5) [39]. This potent M. maydis antifungal compound is se-creted by Trichoderma asperellum (P1), an endophyte isolatedin our laboratory from maize seeds of a cultivar susceptible to LWD [40]. In a follow-up work T. asperellum (P1), or the purified 6-PPmetabolite, significantly improved the infected sprouts and mature plants’ growth parameters and reduced M.maydis DNA in their tissues([40] and unpublished fol-low-up work data). The application of this endophytic species also excels in the field over an entire growing season [38]. At the season’s end, the T. asperellum treatment resulted in 1.6- and 1.3-fold improvement in the LWD symptoms in the lower stem and cob, respec-tively. Furthermore, this treatment led to 4.9-fold lower M. maydis DNA levels in the plants.

Figure 5.Examination and identification of Trichoderma asperellum (P1) active ingredient using GC-MS analysis.The potent M. maydis antifungal metabolite6-pentyl-α-pyrone is secreted by P1, an endophyte isolatedin our laboratory from maize seeds of a cultivar susceptible to LWD [40].This purified active compound was tested in a bioassay in solid growth medium cultures and seeds (adapted from [39]).

Another bio-control approach tested by usismanipulating the plant microbiome.At penetrating the host plant, M. maydismust interact with the maize endophytes, which may provide the plant’s first defense line. Recently, such endophytes were isolated from six sweet and fodder maize hybrids with different sensitivity to LWD[40]. Enriching seeds with two of them, Chaetomium subaffine or T. asperellum,significantly promoted the infected plants’ growth parameters 42 days past sowing. The fungal species Chaetomium cochliodes, T. asperellum, Penicillium citrinum, and the bacteria B. subtilis treatments reduced the LWD pathogen DNA in the host plant’s roots [40].

Finally, preserving soil mycorrhizal fungi between growth periods for crop protection was evaluated[26]. When maize was seeded afterwheat cropping, a significant improve-ment in the shoot’s fresh weight (47–54%) and cob (36–46%) was achieved compared to the other treatments (clover soil, commercial mycorrhiza preparation, and bare soil con-trol). This achievement was not affected drastically by tillage. It was followed by a sharp decrease in disease symptoms (73%) and the pathogen’s presence (82–64%) in the plants’ tissues. It was concluded that since wheat and maize are more closely related (they are both Poaceae) than clover and maize, they might share similar mycorrhizal networks adapted to perform better with these crops.
3. Future Challenges and Opportunities

The late wilt of maize is a challenging disease that imposes a significant economic price in infected areas. Besidesusing resistance germlineand the available chemical option to control LWD,future efforts should focus on searching for new, hazard-free chemicals that are highly effective against M. maydis. One option is combining chemical and biolog-ical approaches [41]. This solution has been proposed to reduce fungicide doses andthe selection pressure on pathogensthat lead toresistance development. Another approach is the development of new eco-friendly options with improved protocols[19]. Maximizing the

efficacy of Trichoderma against M. maydis using freshwater microalgae extractsis an example that can open the door for many similar solutions to LWD.

Bio-friendly protective microorganisms that produce secondary metabolites such as6-pentyl-α-pyrone (6-PP) [39], with powerful activity against M. maydis,can be isolated fromthe maize plants themselves [40]. The roots or seeds of maize plants (apparently LWD-susceptible cultivars are preferred) are inhabited by many beneficial fungi and bac-teria that shield the plant from outside invading pathogens. Identification of these mem-bers of the plant microbiome and exploring their potential may open a vast array of new possibilities to control M. maydis.

*Plant Sciences Department, MIGAL—Galilee Research Institute, Tarshish 2, Kiryat Shmona 11016, Israel;
**Faculty of Sciences, Tel-Hai College, Upper Galilee, Tel-Hai 12210, Israel