Methods for Studying Magnaporthiopsis maydis, the Maize Late Wilt Causal Agent

Abstract

Late wilt, a destructive vascular disease of maize caused by the fungus Magnaporthiopsis maydis, is characterized by relatively fast wilting of maize plants closely before the physiological maturity stage. Previously, traditional microbiology-based methods have been used to isolate the pathogen and to characterize its traits. More recently, several molecular methods have been developed, enabling accurate and sensitive examination of the pathogen spread within the host. Here, we review the methods developed in the past 10 years in Israel, which include new or modified microbial and molecular techniques to identify, monitor, and study M. maydis in controlled environments and in the field. The assays inspected are exemplified with new findings and include microbial isolation methods, microscopic and PCR or qPCR identification, spore germination evaluation, root pathogenicity assay, M. maydis hyphae or filtrate effects on grain germination and sprout development, and a field assay. These diagnostic protocols enable rapid and reliable detection and identification of the pathogen in plants and seeds and studying the pathogenesis of M. maydis in susceptible and relatively resistant maize cultivars in a contaminated field. Moreover, these techniques are important for studying the population structure, and for future development of new strategies to restrict the disease's outburst and spread.The fungus reproduces asexually, and no perfect stage has been identified [5]. To date, late wilt has been reported in about 10 countries, with significant economic losses in Egypt [6], India [7], Spain and Portugal [8], and Israel [9]. In Israel, the sweet maize growth area covered about 2800 ha and yielded almost 57,000 metric tons of grains (data from the Israel Organization of Crops and Vegetables, 2017). Late wilt is considered to be the most destructive disease in maize-growing areas in this country, with up to 100% infection and total yield loss reported in some fields [10]. In Egypt, the cultivated maize area covered about 880,000 ha and yielded almost 7.2 million metric tons of grains [11], with a degree of loss that may reach up to 80% in infested fields [12]. The Egyptian, Indian, and Hungarian isolates of M. maydis differ in morphology, pathogenicity, and route of infection [13]. For example, in Egypt, four clonal lineages of M. maydis isolates show diversity in amplification fragment length polymorphism (AFLP), colonization ability, and virulence on maize [14][15][16][17][18]. In Spain and southern Portugal, 14 isolates of M. maydis were analyzed by inoculating maize-susceptible cultivars [19]. One of the isolates was more aggressive, caused significant weight reductions of both roots and above-ground parts.Late wilt disease is characterized by relatively burst-wilting symptoms of maize plants, typically two weeks after the R1 fertilization growth stage (70 days after sowing) at the R3 growth stage (corn development described by [20]). The pathogen can negatively affect seedling emergence and cause seedling root necrosis...

Isolation and Identification of Fusarium spp., the Causal Agents of Onion (Allium cepa) Basal Rot in Northeastern Israel

“…The onion seedling and bulb pathogenicity assay that was adjusted and used in the current work may be useful for breeding onion cultivars resistance and for screening fungicides, as was done in other crop fungal diseases (see, for example, [31]). Also, this assay may enable us to examine other treatments and to study the penetration and establishment stages of the pathogenesis.…”

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

“…Emergence percentages evaluation conducted 9 DAS for all the plants. The growth parameters were evaluated at the maize sprouting phase (29,(34)(35)(36)(37)54 DAS). Later, at the end of the experiment, the maize harvest day (79-82 DAS), phenological stage evaluation, wilt determination, and yield assessment were performed.…”

“…Tissue samples were placed in universal extraction bags (Bioreba, Reinach, Switzerland) with 4 mL of cetyl hexadecyltrimethylammonium bromide (CTAB) buffer and were ground with a manual tissue homogenizer (Bioreba, Reinach, Switzerland) for 5 min until the tissues were completely homogenized. The homogenized samples were treated for DNA purification as previously described [36]. The DNA was suspended in 100 µL of HPLC-grade water and kept at −20 • C until use in the qPCR analysis.…”

“…All of the qPCR reactions were performed as previously described [36] using the ABI PRISM ® 7900 HT Sequence Detection System (Applied Biosystems, Foster City, CA, USA) for 384-well plates. The 5 µL of total reaction was used per sample well, including 2 µL of sample DNA extract, 2.5 µL of iTaq™ Universal SYBR ® Green Supermix (Bio-Rad Laboratories Ltd., Rishon Le Zion, Israel), 0.25 µL of forward primer and 0.25 µL of reverse primer (10 µM from each primer to a well).…”

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

“…As leaf wilting advances, yellowish or reddish-brown streaks appear on the basal internodes of the stalk, which dries up and shrinks [9]. Due to the delay in appearance of initial symptoms until about flowering, this disease has been designated as "late wilt" [10,11]. M. maydis is a soilborne vascular pathogen that penetrates the root tissues and colonizes the xylem [12].…”