1. Introduction
The importance of maize (
Zea mays L., corn) cannot be over-emphasized, especially in developing countries [
1]. Maize is produced annually more than any other grain, reflecting its importance globally [
2]. Late wilt, a disease severely affecting maize fields throughout Israel [
3], is characterized by rather rapid dehydration of the plants near maturity. It is considered the most harmful disease in commercial maize fields in Israel [
4] and Egypt [
5] and poses a significant threat in India [
6,
7], Spain, and Portugal [
8]. The disease is gradually continuing to spread and is currently reported in at least eight countries. The causal agent is the fungus
Magnaporthiopsis maydis [
9], recognized by two additional synonyms,
Cephalosporium maydis [
10] and
Harpophora maydis [
11,
12].
The pathogen can survive in the soil for long periods. When a susceptible host plant is seeded, the fungi can penetrate the plants’ roots, causing root necrosis and affecting sprout development [
13,
14]. First aboveground symptoms usually appear later in the season as plants begin to flower and are enhanced under drought conditions [
15,
16]. When the growth session advances,
M. maydis spreads upwards inside the plants’ vascular system, disrupts the water supply and leads to dehydration [
17]. In heavily infested fields planted with sensitive maize hybrids, late wilt may cause 100% infection and total yield loss [
18]. If ears are produced, the kernels that do form are poorly developed and are infested with the pathogen.
M. maydis can survive and spread through seeds [
19], infested soil, crop residues [
20], or secondary hosts such as lupine [
21], cotton [
22,
23], watermelon, and
Setaria viridis (green foxtail) [
24]. Various LWD prevention methods have been examined over the years, and some gained positive results in reducing LWD in commercial fields. These include balanced soil fertility [
25,
26], adjusted tillage system and cover crop [
27], watering the field [
28], biological approaches (will be discussed in detail below), soil solarization [
29], allelochemical [
13], and chemical options [
4,
30,
31,
32,
33]. However, none of these methods is currently being used in Israel. Instead, worldwide LWD is controlled by more economically effective management by developing genetically resistant maize cultivars [
5,
6,
34].
The National Maize Program in the Agricultural Research Center in Giza, Egypt, identified many sources of resistance; the release of resistant cultivars since 1980 has significantly reduced late wilt losses in Egypt [
35]. A breeding program for resistant germ lines has been operational in Israel for about a decade (Israel Northern R&D, Migal–Galilee Research Institute, Kiryat Shmona, Israel). However, the presence of highly aggressive isolates of
M. maydis [
36,
37] may threaten these resistant maize cultivars. In addition, the pathogen could spread in relatively resistant plants that showed no symptoms. Infected seeds, even those of non-symptomatic plants, can spread the disease [
3,
19].
In 2017–2018, the search for a chemical application to control LWD led to an economically feasible solution [
4,
18,
31] in Israel. The successful treatment protocol is based on changing the maize cultivation method, changing the traditional irrigation method used in most corn-growing areas in Israel, and the sophisticated integration of Azoxystrobin-based pesticide mixtures in a schedule adapted to key points in the development of the disease.
Still, it was shown earlier that the pathogen is present in the host tissues of successfully chemically treated plants [
32]. This finding hints at the potential risk that the pathogen will develop immunity to Azoxystrobin, the most effective antifungal compound against the late wilt pathogen [
4,
18]. Unfortunately, the rapid development of resistance to this fungicide and the consequential control failure in many crops has become increasingly problematic [
38].
The growing trend of reducing pesticide use [
39] raises the need for alternative ways of coping with severe fungal diseases such as the late wilt of maize. Indeed, biological eco-friendly control methods to restrict
M. maydis and other phytopathogens are at the forefront of the current scientific effort. Two environmentally friendly strategies to control late wilt are presently in this scientific focus.
First, the use of
Trichoderma sp. and other protective microorganisms as a biocontrol agent has been demonstrated in the past in culture media, in greenhouse plants, and in the field with very promising potential (most recently [
40,
41]). Likewise, we previously conducted two years of research with new
Trichoderma species:
T. asperelloides (T.203);
T. longibrachiatum (T.7407 from marine source [
42]); and
T. asperellum (P1), an endophyte isolated in our laboratory from corn seeds of a strain susceptible to LWD [
43]. These isolates prevented the pathogen’s growth in culture plates, significantly reduced its establishment and development in seedlings’ corn plant tissues, and resulted in significant improvement in growth and crop indices under field conditions [
16,
44].
Second, maintaining soil mycorrhizal fungi between seasons has proven to be an essential factor towards the same goal (summarized by [
40]). Results from other phytopathogens suggest that under no-till cropping, selected cover crops or crops in a rotation could help build mycorrhizal communities that function throughout a sequence of several main crops [
45]. Aggressive tillage combined with long periods where the field is unprocessed results in the destruction of the integrity of mycorrhizal networks. Maintaining the continuity and integrity of mycorrhizal networks in the soil may allow the plant to enjoy higher resistance to soil diseases [
46], including late wilt disease [
27]. Indeed, Arbuscular mycorrhizal fungi (AMF) have a proven ability to improve plant resistance to biotic and abiotic stresses by activated the plant’s local and systemic defense mechanisms [
47].
So far, this approach of strengthening the soil microbiome was poorly tested against the late wilt pathogen,
M. maydis. This knowledge gap is now encouraging the exploration of this method’s potential as part of an integrated control program to restrain the late wilt agent, as we will detail below. In the current study, this method was studied thoroughly and evaluated over two years. In the first year, the effect of crop rotations of wheat/maize or clover/maize and a simulation of tillage regimes (no-tillage or conventional tillage) was inspected in pots where LWD-susceptible maize cultivar was grown in infected soil. These treatments were compared to infected no-tillage soil and infected no-tillage soil + commercial mycorrhizal preparation (Resid MG [
48], BioBee, Sde Eliyahu, Israel). In the second year, the field’s soils after commercial cultivation of wheat, clover, or soil without previous winter growth were used. These soils underwent two different tillage regimes before maize cultivation—no-tillage or conventional tillage. The effectiveness of these practices on
M. maydis pathogenesis was studied by evaluating the growth parameters throughout the season, estimating the disease symptoms, measuring yield production, and monitoring the pathogen’s DNA inside the host tissues using quantitative real-time (qPCR)-based approach.
4. Discussion
The rhizosphere, a soil layer adjacent to the root’s surface, is affected by both the presence of a plant and soil properties. This layer has a critical impact on a plant’s existence. A significant part of the roots and the whole plant function depends on the “nature of the rhizosphere” and the biological activities within it. Intensive agriculture causes a decrease in microbial biomass in the soil that causes, over time, a decline in soil fertility and yield [
58]. Arbuscular mycorrhizal fungi (AMF), which interact with plant roots and other soil fungi, have known benefits in nourishing plants and conferring disease resistance. These properties make them a valuable tool in modern agriculture [
48]. The fungus provides the plant with nutrients, affects root morphology, and improves water balance, while the plant provides it with photosynthesis products. These “natural fertilizers” are obligatory symbionts belonging to the phylum
Glomeromycota and inhabit 80% of terrestrial plants [
59]. Mycorrhiza has an aggressive antagonism with various plant pathogens and may also be used as a biological pesticide [
60].
To date, the use of mycorrhizal fungi to protect field crops is limited due to the high costs (compared to chemical fertilizers) and low biodiversity of the commercial applications offered [
59]. This is in addition to the long time required from applying the fungus to achieving efficiency, and in light of the fact that such applications often do not match the intensive growing systems used in agricultural fields. At the same time, mycorrhizal fungi have a wide range of plant hosts. Moreover, using the intact extraradical mycelium method (ERM, preserving the continuity of the mycorrhizal network in the soil) makes it possible to produce a crop sequence that will maintain the mycorrhizal network that promotes the plants’ growth and assist in protecting them against soil diseases [
61].
Although the worldwide scientific effort is focusing on seeking solutions to LWD based on eco-friendly biological approaches [
16,
40,
41,
43,
44,
62], there is a lack of information on maize performance under LWD stress in crop rotation and reduced tillage. One study that examined this recently [
27] showed that grain production and
M. maydis presence were significantly reduced when both cover crop and minimum tillage were applied together. It was also found that with cover crop and minimum tillage, the arbuscular root colonization was higher.
The current study did not directly examine the effect of preserving and establishing the mycorrhizal network in the soil to deal with late wilt disease in corn. Instead, it focused, for the first time in Israel, on an agricultural practice based on preserving soil microflora integrity (by avoiding tillage) and affecting its nature (by cultivating selected crops in a dual-season growth). A comparative analysis of the 2019 and the 2020 experiments results at the harvest day is illustrated in
Table 7.
When corn was sown on wheat soil, a significant improvement in the fresh weight of the shoot (147–154% in the 2019 experiment) and cob (136–146% in the 2019 experiment) was achieved compared to the control. This result was also better than the other treatments (clover soil and commercial mycorrhiza preparation). This achievement was not affected drastically by tillage. It was followed by a sharp decrease in disease symptoms (73% in the 2019 experiment) and the pathogen’s presence (82–64% in the 2019 experiment) in the plants’ tissues (
Table 8).
Interestingly, the 2020 experiment that was based on commercial fields soil resulted in more severe LWD. The disease severity is reflected in higher diseased plants percentages and sharp (23-fold) elevation in the pathogen DNA in the first aboveground internode of the no-tillage control plants (
Figure 7,
Table 8). This is most likely the reason for the lesser enhancement in the shoot and cobs fresh weight in the clover and wheat treatments (compared to the control) this year (
Table 7).
Another intriguing aspect is that at the season-ending, the roots appeared comparatively less affected by the pathogen than the shoot (
Figure 7). Indeed,
M. maydis high DNA levels in the shoot at this growth stage (the harvest) are well documented in our previous works [
3,
4]. When an LWD susceptible maize cultivar was seeded on
M. maydis heavily infested commercial field soil, the following changes in the pathogen’s DNA levels in the host tissues were recorded. When early signs of the disease began to appear from day 50 onwards, the fungal DNA decreased in the roots but increased significantly in the stems until it peaked at 3.2-fold its initial level 72 d after sowing [
3]. These variations in the DNA were consistent with previous literature observations on the disease’s mode and the pathogen spread from the roots to the stem, leaves, and kernels [
63].
Thus, it appears that since wheat and maize are more closely related than clover and maize (they are both Poaceae), they may share similar mycorrhizal networks that are adapted to perform better with these crops. Indeed, in the clover–maize sequence, the above-mentioned growth promotion and LWD resistance were reduced, even more so when tillage was applied.
To support this idea, it was reported that crop plants acquired a mycorrhizal fungal community closely related to that of the previous host plant and different from that found when the soil was disturbed or not cropped before growth [
45]. In this last study, wheat grown in undisturbed soil immediately after the legume
Ornithopus compressus acquired a mycorrhizal fungal community closely related to that of the last plant host and different from that found when the soil was disturbed or not cropped prior to the growth of the wheat. Parallel effects were seen in the succession from Poaceae (
Lolium rigidum) to Fabaceae (
Trifolium subterraneum), indicating that these effects are not unique to the legume-wheat sequence.
With the novelty of the current study, it is essential to point out that these trials were conducted over one season, while crop rotation may have a long-term effect on soil fungus populations, which may only be evident after a more extended period. A long-term study on the impact of different crop rotations (including wheat–maize) and tillage regimes (no-tillage or conventional tillage) on microbial biomass and other soil properties was established in 1976 in southern Brazil [
58]. The no-tillage system showed increases in microbial biomass percentage at the 0- to 5-cm depth. Reduction of tillage had a more significant effect on microbial biomass than crop rotations, particularly in the 0–5 cm depth. These results provide evidence that tillage or crop rotation affects microbial immobilization of soil nutrients. The results of this and other studies indicate that no single cropping system is favored for all fungi [
64]. Thus, a tailored solution that will address the crops in the rotation, the tillage system, the cultivar that should be protected, and the pathogens/disease stresses should be carefully planned. Such situations should be examined in subsequent studies. Closely related cultivars such as wheat and maize may benefit from the same control strategy.