Siderophore production by Bacillus subtilis MF497446 and Pseudomonas koreensis MG209738 and their efficacy in controlling Cephalosporium maydis in maize plant.

Late wilt disease, caused by Cephalosporium maydis in maize plant, is one of the main economical diseases in Egypt. Therefore, to cope with this problem, we investigated the potentiality of plant growth promoting rhizobacteria in controlling this disease. Six strains (Bacillus subtilis, B. circulance, B. coagulanse, B. licheniformis, Pseudomonas fluroscence and P. koreensis) were screened for siderophore production, and using dual plate culture method and greenhouse experiment, antagonistic activity against C. maydis was studied. Using two superior strains, single and dual inoculation treatments in maize were applied in field experiment during the 2018 and 2019 seasons. Results indicated that B. subtilis and P. koreensis strains had shown the most qualitative and quantitative assays for siderophore production and antagonistic activities. In greenhouse, the most effective treatments on the pre- and post-emergence damping off as well as growth promotion of maize were T3 treatment (inoculated with B. subtilis), and T8 treatment (inoculated with P. koreensis). In field experiment, T5 treatment (inoculated with a mixture of B. subtilis and P. koreensis) showed significant increases in catalase (CAT), peroxidase (POX) and polyphenol oxidase (PPO) activities, as well as total chlorophyll and carotenoids than control treatments during the two growing seasons. In the same way, the highest effect in reducing infection and increasing the thickness of the sclerenchymatous sheath layer surrounding the vascular bundles in maize stem was observed and these results were a reflection of the increase in yield and yield parameters.

Plant growth hormones suppress the development of Harpophora maydis, the cause of late wilt in maize.

Late wilt, a severe vascular disease of maize caused by the fungus Harpophora maydis, is characterized by rapid wilting of maize plants before tasseling and until shortly before maturity. The pathogen is currently controlled by resistant maize cultivars, but the disease is constantly spreading to new areas. The plant's late phenological stage at which the disease appears suggests that plant hormones may be involved in the pathogenesis. This work revealed that plant growth hormones, auxin (Indole-3-acetic acid) and cytokinin (kinetin), suppress H. maydis in culture media and in a detached root assay. Kinetin, and even more auxin, caused significant suppression of fungus spore germination. Gibberellic acid did not alter colony growth rate but had a signal suppressive effect on the pathogens' spore germination. In comparison, ethylene and jasmonic acid, plant senescing and defense response regulators, had minor effects on colony growth and spore germination rate. Their associate hormone, salicylic acid, had a moderate suppressive effect on spore germination and colony growth rate, and a strong influence when combined with auxin. Despite the anti-fungal auxin success in vitro, field experiments with dimethylamine salt of  2,4-dichlorophenoxyacetic acid (that mimics the influence of auxin) failed to suppress the late wilt. The lines of evidence presented here reveal the suppressive influence of the three growth hormones studied on fungal development and are important to encourage further and more in-depth examinations of this intriguing hormonal complex regulatory and its role in the maize-H. maydis interactions.

Discovery of genomic regions associated with resistance to late wilt disease caused by Harpophora maydis (Samra, Sabet and Hing) in maize (Zea mays L.).

Late wilt disease (LWD) caused by Harpophora maydis (Samra, Sabet and Hing) is emerging as major production constraint in maize across the world. As a prelude to develop maize hybrid resistance to LWD, genetic basis of resistance was investigated. Two F2:3 mapping populations (derived from CV156670 × 414-33 (P-1) and CV156670 × CV143587 (P-2)) were challenged with LWD at two locations (Kallinayakanahalli and Muppadighatta) during 2017 post-rainy season. A wider range of LWD scores was observed at both locations in both the populations. LWD response was influenced by significant genotype × location interaction. Six and 56 F2:3 progeny families showed resistance level better than resistant parent. A total of 150 and 199 polymorphic single nucleotide polymorphism markers were used to genotype P-1 and P-2, respectively. Inclusive composite interval mapping was performed to detect significant Quantitative Trait Loci (QTL), QTL × QTL, QTL × location interaction effects. Three major and four minor QTL controlling LWD resistance were detected on chromosome-1. The position and effect of the QTL varied with the location. Significant di-QTL interactions involving QTL (with significant and/or non-significant effects) located within and between all the ten chromosomes were detected. Five of the seven detected QTL showed significant QTL × location interaction. Though two major QTL (q-lw-1.5 and q-lw-1.6) with lower Q×L interaction effects could be considered as stable, their phenotypic variance is not large enough to deploy them in Marker Assisted Selection (MAS). However, these QTL are of paramount importance in accumulating positive alleles for LWD resistance breeding.

Cultivars Resistance Assay for Maize Late Wilt Disease.

Magnaporthiopsis maydis late wilt disease (LWD) in corn is considered to be the most severe in Israel and Egypt and poses a significant threat in other countries. Research efforts extending over a period of five decades led to the development of chemical, biological, agrotechnical, physical (solar disinfection) and other means for controlling late wilt disease. Today, some applications can reduce damage even in severe cases. However, cultivating disease-resistant maize varieties is the primary means for reducing the disease's impact. The current work uses a rapid (six days) laboratory seedling pathogenicity test and a full-season open encloser semi-field conditioned pots assay (101 days) to classify maize varieties according to their LWD resistance. To better evaluate differences between the cultivars, a real-time based molecular assay was applied to track the pathogen's presence in the plants' tissues, and visible light aerial imaging was used in parallel. The findings show that in cases of extreme sensitivity or tolerance (for example, in the highly susceptible Megaton cultivar (cv.) or the resistant Hatai cv.), a similarity in the results exists between the different methods. Thus, a reliable estimate of the varieties' sensitivity can be obtained in a seed assay without the need for a test carried out throughout an entire growing season. At the same time, in most situations of partial or reduced LWD sensitivity/resistance, there is no match between the various tests, and only the entire growing season can provide the most reliable results. Tracking the amount of M. maydis DNA in the plants' bodies is a precise, sensitive scientific tool of great importance for studying the development of the disease and the factors affecting it. Yet, no complete overlap exists between the fungal DNA amount and symptom severity. Such a correlation exists in high sensitivity or resistance cases but not in intermediate situations. Still, the valuation of the pathogen's establishment in asymptomatic corn hybrids can indicate the degree of LWD immunity and the chance of susceptibility development.

Aggressive strains of the late wilt fungus of corn exist in Israel in mixed populations and can specialize in disrupting growth or plant health.

Maize late wilt disease caused by the fungus Magnaporthiopsis maydis significantly damages crops in Israel and in other countries. Resistant maize cultivars are the preferred method for disease restraining. However, the pathogen populations of Spain and Egypt have varying aggressiveness, and virulent strains can overcome host resistance. In 2001 and from 2016 to -2019, 17 M. maydis strains were isolated from infected maize fields in Israel. The isolates' effects on seed germination, plant development, and disease symptoms severity were evaluated. The isolates from Israel display a diverse degree of aggressiveness that is not linked to their geographic distribution. The virulent strains are found in mixed populations, whereas less virulent M. maydis isolates exist. Aggressive strains harmed the development of plants and ears and caused severe wilting and death. In contrast, plants inoculated with less virulent strains exhibited only mild dehydration signs, and crop yield was similar to that of the non-infected control. Interestingly, different host cultivars can evoke specific virulence of M. maydis strains. Moreover, some pathogen strains significantly repress plant development, while the impact of other strains was evidenced by wilting symptoms. The current research further increases our understanding of the pathogen and our ability to control it.

Crop Rotation and Minimal Tillage Selectively Affect Maize Growth Promotion under Late Wilt Disease Stress.

In recent years, worldwide scientific efforts towards controlling maize late wilt disease (LWD) have focused on eco-friendly approaches that minimize the environmental impact and health risks. This disease is considered to be the most severe threat to maize fields in Israel and Egypt, and a major growth restraint in India, Spain, and Portugal. Today's most commonly used method for LWD control involving resistant maize genotypes is under constant risk from aggressive pathogen lines. Thus, this study's objectives were to evaluate the effect of crop rotation and avoiding tillage on restraining the disease. Such an agrotechnical approach will support the continuity of soil mycorrhiza networks, which antagonize the disease's causal agent, Magnaporthiopsis maydis. The method gained positive results in previous studies, but many knowledge gaps still need to be addressed. To this end, a dual-season study was conducted using the LWD hyper-susceptible maize hybrid, Megaton cv. The trials were performed in a greenhouse and in the field over full dual-growth seasons (wheat or clover as the winter crop followed by maize as the summer crop). In the greenhouse under LWD stress, the results clearly demonstrate the beneficial effect of maize crop rotation with clover and wheat on plant weight (1.4-fold), height (1.1-1.2-fold) and cob yield (1.8-2.4-fold), especially in the no-till soil. The clover-maize growth sequence excels in reducing disease impact (1.7-fold) and pathogen spread in the host tissues (3-fold). Even though the wheat-maize crop cycle was less effective, it still had better results than the commercial mycorrhizal preparation treatment and the uncultivated non-infected soil. The results were slightly different in the field. The clover-maize rotation also achieved the best growth promotion and disease restraint results (2.6-fold increase in healthy plants), but the maize rotation with wheat showed only minor efficiency. Interestingly, pre-cultivating the soil with clover had better results in no-till soil in both experiments. In contrast, the same procedure with wheat had a better impact when tillage was applied. It may be concluded that crop rotation and soil cultivation can be essential in reducing LWD, but other factors may affect this approach's benefits in commercial field growth.

Crop Cycle and Tillage Role in the Outbreak of Late Wilt Disease of Maize Caused by Magnaporthiopsis maydis.

The destructive maize late wilt disease (LWD) has heavy economic implications in highly infected areas such as Israel, Egypt, and Spain. The disease outbreaks occur near the harvest, leading to total yield loss in severe cases. Crop rotation has long been known as an effective means to reduce plant diseases. Indeed, agricultural soil conservation practices that can promote beneficial soil and root fungi have become increasingly important. Such methods may have a bioprotective effect against Magnaporthiopsis maydis, the LWD causal agent. In this two-year study, we tested the role of crop rotation of maize with either wheat or clover and the influence of minimum tillage in restricting LWD. In the first experiment, wheat and clover were grown in pots with LWD infected soil in a greenhouse over a full winter growth period. These cultivations were harvested in the spring, and each pot's group was split into two subgroups that underwent different land processing practices. The pots were sown with LWD-sensitive maize cultivar and tested over a whole growth period against control soils without crop rotation or soil with commercial mycorrhizal preparation. The maize crop rotation with wheat without tillage achieved prominent higher growth indices than the control and the clover crop cycle. Statistically significant improvement was measured in the non-tillage wheat soil pots in sprout height 22 days after sowing, in the healthy plants at the season's end (day 77), and in shoot and cob wet weight (compared to the control). This growth promotion was accompanied by a 5.8-fold decrease in pathogen DNA in the plant stems. The tillage in the wheat-maize growth sequence resulted in similar results with improved shoot wet-weight throughout the season. In contrast, when maize was grown after clover, the tillage reduced this parameter. The addition of commercial mycorrhizal preparation to the soil resulted in higher growth measures than the control but was less efficient than the wheat crop cycle. These results were supported by a subsequent similar experiment that relied on soil taken from commercial wheat or clover fields. Here too, the wheat-maize growth cycle (without permanent effect for the tillage) achieved the best results and improved the plants' growth parameters and immunity against LWD and lowered pathogen levels. In conclusion, the results of this study suggest that wheat and perhaps other crops yet to be inspected, together with the adjusted tillage system, may provide plants with better defense against the LWD pathogen.

Effect of Tillage System and Cover Crop on Maize Mycorrhization and Presence of Magnaporthiopsis maydis.

The sustainability of agriculture requires the adoption of agricultural soil conservation practices with positive impacts on soil quality, which can promote beneficial soil microbiota like arbuscular mycorrhizal fungi (AMF) and its diversity. This study aims to assess the influence of the presence of intact extraradical mycelium as a preferential source of inoculum of the native AMF in order to guarantee a better colonization as well as its possible bioprotective effect against Magnaporthiopsis maydis. In order to vary the available extraradical mycelium, two experiments, with and without cover crop, were carried out, in which two tillage systems and two maize varieties were studied. The capitalization of the benefits, in terms of grain production and M. maydis presence, associated to the cover crop were only achieved with minimum tillage. Therefore, both cultural practices are necessary to reduce the fungus presence, coupling the effect of mycorrhization together with other benefits associated with the cover crop. Although in the absence of a cover crop and using conventional tillage, yields and lower levels of M. maydis are possibly achieved, this system is more dependent on the variety used, does not benefit from the advantages associated with the cover crop, is more expensive, and environmentally unsustainable.

Soil Bioassay for Detecting Magnaporthiopsis maydis Infestation Using a Hyper Susceptible Maize Hybrid.

Magnaporthiopsis maydis is the causal agent of severe maize late wilt disease. Disease outbreak occurs at the maize flowering and fruit development stage, leading to the plugging of the plant's water vascular system, resulting in dehydration and collapse of the infected host plant. The pathogen is borne by alternative hosts, infected seeds, soil, and plant residues and gradually spreads to new areas and new countries. However, no soil assay is available today that can detect M. maydis infestation and study its prevalence. We recently developed a molecular quantitative Real-Time PCR (qPCR) method enabling the detection of the M. maydis DNA in plant tissues. Despite the technique's high sensitivity, the direct examination of soil samples can be inconsistent. To face this challenge, the current work demonstrates the use of a soil bioassay involving the cultivation of a hyper-susceptible maize genotype (Megaton cultivar, Hazera Seeds Ltd., Berurim MP Shikmim, Israel) on inspected soils. The use of Megaton cv. may facilitate pathogen establishment and spread inside the plant's tissues, and ease the isolation and enrichment of the pathogen from the soil. Indeed, this cultivar suffers from severe dehydration sudden death when grown in an infested field. The qPCR method was able to accurately and consistently identify and quantify the pathogen's DNA in an in vitro seed assay after seven days, and in growth-chamber potted plants at as early as three weeks. These results now enable the use of this highly susceptible testing plant to validate the presence of the maize late wilt pathogen in infested soils and to evaluate the degree of its prevalence.

Interactions between Magnaporthiopsis maydis and Macrophomina phaseolina, the Causes of Wilt Diseases in Maize and Cotton.

Fungal pathogens are a significant threat to crops worldwide. The soil fungus, Magnaporthiopsis maydis, severely affects sensitive maize hybrids by causing the rapid wilting of plants at the maturity stage. Similarly, the soil fungus, Macrophomina phaseolina, develops in a variety of host plants, which leads to rot and plant mortality. The presence of both pathogens together in diseased cotton plants in Israel suggests possible interactions between them. Here, these relationships were tested in a series of experiments accompanied by real-time PCR tracking in maize and cotton. Despite the fact that neither of the pathogens was superior in a growth plate confrontation assay, their co-inoculum had a significant influence under field conditions. In maize sprouts and fully matured plants, infection by both pathogens (compared to inoculation with each of them alone) led to lesser amounts of M. maydis DNA but to increased amounts of M. phaseolina DNA levels. These results were obtained under a restricted water regime, while optimal water irrigation led to less pronounced differences. In water-stressed cotton sprouts, infection with both pathogens led to an increase in DNA amounts of each of the pathogens. Whereas the M. maydis DNA levels in the double infection remain high at the end of the season, a reduction in the amount of M. phaseolina DNA was observed. The double infection caused an increase in growth parameters in maize and cotton and decreased levels of dehydration in maize plants accompanied by an increase in yield production. Dehydration symptoms were minor in cotton under an optimal water supply. However, under a restricted water regime, the double infection abolished the harmful effect of M. phaseolina on the plants' development and yield. These findings are the first report of interactions between these two pathogens in maize and cotton, and they encourage expanding the study to additional plant hosts and examining the potential involvement of other pathogens.

Uncovering the Host Range for Maize Pathogen Magnaporthiopsis maydis.

The fungus Magnaporthiopsis maydis is a soil-borne, seed-borne vascular wilt pathogen that causes severe damage to sensitive Zea mays L. (maize) hybrids throughout Egypt, Israel, India, Spain, and other countries. It can undergo virulence variations and survive as spores, sclerotia, or mycelia on plant residues. Maize, Lupinus termis L. (lupine) and Gossypium hirsutum L. (cotton) are the only known hosts of M. maydis. Identification of new plant hosts that can assist in the survival of the pathogen is an essential step in restricting disease outbreak and spread. Here, by field survey and growth chamber pathogenicity test, accompanied by real-time PCR analysis, the presence of the fungal DNA inside the roots of cotton (Pima cv.) plants was confirmed in infested soil. Moreover, we identified M. maydis in Setaria viridis (green foxtail) and Citrullus lanatus (watermelon, Malali cv.). Infected watermelon sprouts had delayed emergence and development, were shorter, and had reduced root and shoot biomass. M. maydis infection also affected root biomass and phenological development of cotton plants but caused only mild symptoms in green foxtail. No M. maydis DNA was detected in Hordeum vulgare (barley, Noga cv.) and the plants showed no disease symptoms except for reduced shoot weight. These findings are an important step towards uncovering the host range and endophytic behavior of M. maydis, encouraging expanding this evaluation to other plant species.

Comparison of Gaeumannomyces- and Phialophora-like fungal pathogens from maize and other plants using DNA methods.

Several DNA-based techniques, developed for identifying and differentiating fungi in the Gaeumannomyces-Phialophora complex associated with take-all diseases of cereals and grasses, were used to compare fungi from maize. Maize isolates obtained as G. graminis (Sacc.) Arx & H Olivier var. tritici Walker, from the UK, having been identified by ascospore morphology and in pathogenicity tests on wheat, were indistinguishable from isolates of the same variety obtained from wheat. Isolates of G. graminis (Sacc.) Arx & H Olivier var. maydis Yao et al., recently described as the maize take-all fungus from China, were identical in DNA tests to the anamorphic fungus Phialophora radicicola Cain and almost identical to Phialophora zeicola Deacon & Scott, whose description was originally based on isolates from South Africa and France. These species appear to represent the holomorph of the same fungus. The late wilt pathogen of maize, from India and Egypt, commonly known as Cephalosporium maydis Samra et al., but suggested as being the Phialophora anamorph of a Gaeumannomyces species, was closely related to other Gaeumannomyces species included in the tests.

Relative Competitiveness and Virulence of Four Clonal Lineages of Cephalosporium maydis from Egypt Toward Greenhouse-Grown Maize.

Four clonal lineages of Cephalosporium maydis, a soilborne vascular wilt pathogen that causes late wilt of maize, were differentiated previously with molecular markers. In Egypt, this fungus can cause significant losses in infected susceptible plants. In greenhouse tests of individual isolates we found that these lineages differ in their virulence toward a series of maize accessions commonly used in Egyptian maize breeding programs. We also determined the relative competitiveness of representatives of the four lineages when incorporated into the soil as a mixed inoculum. The lineage (IV) with greatest mean disease incidence (virulence), when tested alone, was the least competitive on susceptible maize accessions when coinoculated as a component of mixed inocula of all four lineages. In these coinoculation experiments, one of the less-virulent lineages (II) dominated (70% of infections) and appeared to be the most competitive. These results suggest that virulence and competitive ability are not the same in this host-pathogen system. These results also suggest that standard protocols that rely on mixed inocula for resistance screening need to be altered, and that the relative proportion of the different lineages of the pathogen recovered in a field may be influenced by the maize variety/hybrid planted.