Integrated Management of the Cotton Charcoal Rot Disease Using Biological Agents and Chemical Pesticides.

Charcoal rot disease (CRD), caused by the phytopathogenic fungus Macrophomina phaseolina, is a significant threat to cotton production in Israel and worldwide. The pathogen secretes toxins and degrading enzymes that disrupt the water and nutrient uptake, leading to death at the late stages of growth. While many control strategies were tested over the years to reduce CRD impact, reaching that goal remains a significant challenge. The current study aimed to establish, improve, and deepen our understanding of a new approach combining biological agents and chemical pesticides. Such intervention relies on reducing fungicides while providing stability and a head start to eco-friendly bio-protective Trichoderma species. The research design included sprouts in a growth room and commercial field plants receiving the same treatments. Under a controlled environment, comparing the bio-based coating treatments with their corresponding chemical coating partners resulted in similar outcomes in most measures. At 52 days, these practices gained up to 38% and 45% higher root and shoot weight and up to 78% decreased pathogen root infection (tracked by Real-Time PCR), compared to non-infected control plants. Yet, in the shoot weight assessment (day 29 post-sowing), the treatment with only biological seed coating outperformed (p < 0.05) all other biological-based treatments and all Azoxystrobin-based irrigation treatments. In contrast, adverse effects are observed in the chemical seed coating group, particularly in above ground plant parts, which are attributable to the addition of Azoxystrobin irrigation. In the field, the biological treatments had the same impact as the chemical intervention, increasing the cotton plants' yield (up to 17%), improving the health (up to 27%) and reducing M. phaseolina DNA in the roots (up to 37%). When considering all treatments within each approach, a significant benefit to plant health was observed with the bio-chemo integrated management compared to using only chemical interventions. Specific integrated treatments have shown potential in reducing CRD symptoms, such as applying bio-coating and sprinkling Azoxystrobin during sowing. Aerial remote sensing based on high-resolution visible-channel (RGB), green-red vegetation index (GRVI), and thermal imaging supported the above findings and proved its value for studying CRD control management. This research validates the combined biological and chemical intervention potential to shield cotton crops from CRD.

The cotton charcoal rot causal agent, Macrophomina phaseolina, biological and chemical control.

The fungus Macrophomina phaseolina causes charcoal rot disease (CRD) in cotton, whose symptoms develop in the late stages of growth and result in wilting and death. Despite significant research efforts to reduce disease incidences, effective control strategies against M. phaseolina are an ongoing scientific effort. Today's CRD control tends toward green options to reduce the chemicals' environmental footprint and health risks. Here, different Trichoderma species were examined separately and in combination with Azoxystrobin (AS) in semi-field open-enclosure pots and a commercial field throughout a full season. In the pot experiment, the T. asperellum (P1) excelled and led to improvement in growth (13%-14%, day 69) and crops (the number of capsules by 36% and their weight by 78%, day 173). The chemical treatment alone at a low dose had no significant impact. Still, adding AS improved the effect of T. longibrachiatum (T7507) and impaired P1 efficiency. Real-time PCR monitoring of the pathogen DNA in the plants' roots at the harvest (day 176), revealed the efficiency of the combined treatments: T. longibrachiatum (T7407 and T7507) + AS. In a commercial field, seed dressing with a mixture of Trichoderma species (mix of P1, T7407, and Trichoderma sp. O.Y. 7107 isolate) and irrigation of their secreted metabolites during seeding resulted in the highest yields compared with the control. Applying only AS irrigation at a low dose (2,000 cc/ha), with the sowing, was the second best in promoting crops. The molecular M. phaseolina detection showed that the AS at a high dose (4,000 cc/ha) and the biological mix treatments were the most effective. Reducing the AS chemical treatment dosages by half impaired its effectiveness. Irrigation timing, also studied here, is proven vital. Early water opening during the late spring suppresses the disease outburst and damages. The results demonstrated the benefits of CRD bio-shielding and encouraged to explore the potential of a combined bio-chemo pest control approach. Such interphase can be environmentally friendly (reducing chemical substances), stabilize the biological treatment in changing environmental conditions, achieve high efficiency even in severe CRD cases, and reduce the development of fungicide resistance.

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.

Occurrence of Macrophomina phaseolina in Israel: Challenges for Disease Management and Crop Germplasm Enhancement.

Macrophomina phaseolina is a soil-borne fungal pathogen infecting many important crop plants. The fungus, which can survive on crop debris for a long period of time, causes charcoal rot disease by secreting a diverse array of cell-wall degrading enzymes and toxins. M. phaseolina thrives during periods of high temperatures and arid conditions, as typically occur in Israel and other countries with a Mediterranean climate. Crop losses due to charcoal rot can be expected to increase and spread to other countries in a warming global climate. Management of this pathogen is challenging, requiring an array of approaches for the various crop hosts. Approaches that have had some success in Israel include grafting of melons and watermelons on resistant squash rootstocks and soil application of fungicide to reduce disease incidence in melons, fumigation and alterations in planting date and mulching of strawberries, and alteration in irrigation regime of cotton. Elsewhere, these approaches, as well as soil amendments and addition of organisms that are antagonistic to M. phaseolina, have had success in some crop situations. Management through host resistance would be the most sustainable approach, but it requires identifying a resistant germplasm for each crop and introgressing the resistance into the leading cultivars. Resistance to charcoal rot is under complex genetic control in most crops, posing a great challenge for its introgression into elite germplasm. Moreover, fast, reliable methods of screening for resistance would have to be developed for each crop. The toothpick-inoculation method used by us holds great promise for selecting resistant germplasm for melons and possibly for sesame, but other methodologies have to be devised for each individual crop.

Trichoderma longibrachiatum and Trichoderma asperellum Confer Growth Promotion and Protection against Late Wilt Disease in the Field.

Late wilt disease (LWD) of maize, caused by Magnaporthiopsis maydis, is considered a major threat to commercial fields in Israel, Egypt, Spain, and India. Today's control methods include chemical and agronomical intervention but rely almost solely on resistant maize cultivars. In recent years, LWD research focused on eco-friendly biological approaches to restrain the pathogen. The current study conducted during two growing seasons explores the potential of three Trichoderma species as bioprotective treatments against LWD. These species excelled in preliminary assays performed previously under controlled conditions and were applied here in the field by directly adding them to each seed with the sowing. In the first field experiment, Trichoderma longibrachiatum successfully rescued the plants' growth indices (weight and height) compared to T. asperelloides and the non-treated control. However, it had no positive effect on yield and disease progression. In the subsequent season, this Trichoderma species was tested against T. asperellum, an endophyte isolated from susceptible maize cultivar. This experiment was conducted during a rainy autumn season, which probably led to a weak disease burst. Under these conditions, the plants in all treatment groups were vivid and had similar growth progression and yields. Nevertheless, a close symptoms inspection revealed that the T. longibrachiatum treatment resulted in a two-fold reduction in the lower stem symptoms and a 1.4-fold reduction in the cob symptoms at the end of the seasons. T. asperellum achieved 1.6- and 1.3-fold improvement in these parameters, respectively. Quantitative Real-time PCR tracking of the pathogen in the host plants' first internode supported the symptoms' evaluation, with 3.1- and 4.9-fold lower M. maydis DNA levels in the two Trichoderma treatments. In order to induce LWD under the autumn's less favorable conditions, some of the plots in each treatment were inoculated additionally, 20 days after sowing, by stabbing the lower stem section near the ground with a wooden toothpick dipped in M. maydis mycelia. This infection method overrides the Trichoderma roots protection and almost abolishes the biocontrol treatments' protective achievements. This study suggests a biological Trichoderma-based protective layer that may have significant value in mild cases of LWD.

Imazapic Herbigation for Egyptian Broomrape (Phelipanche aegyptiaca) Control in Processing Tomatoes-Laboratory and Greenhouse Studies.

Parasitic plants belonging to the Orobanchaceae family include species that cause heavy damage to crops in Mediterranean climate regions. Phelipanche aegyptiaca is the most common of the Orobanchaceae species in Israel inflicting heavy damage to a wide range of broadleaf crops, including processing tomatoes. P. aegyptiaca is extremely difficult to control due to its minute and vast number of seeds and its underground association with host plant roots. The highly efficient attachment of the parasite haustoria into the host phloem and xylem enables the diversion of water, assimilates and minerals from the host into the parasite. Drip irrigation is the most common method of irrigation in processing tomatoes in Israel, but the delivery of herbicides via drip irrigation systems (herbigation) has not been thoroughly studied. The aim of these studies was to test, under laboratory and greenhouse conditions, the factors involved in the behavior of soil-herbigated imazapic, and the consequential influence of imazapic on P. aegyptiaca and tomato plants. Dose-response Petri dish studies showed that imazapic does not impede P. aegyptiaca seed germination and non-attached seedlings, even at the high rate of 5000 ppb. Imazapic applied to tomato roots inoculated with P. aegyptiaca seeds in a PE bag system revealed that the parasite is killed only after its attachment to the tomato roots, at concentrations as low as 2.5 ppb. Imazapic sorption curves and calculated Kd and Koc values indicated that the herbicide Kd is similar in all soils excluding a two-fold higher coefficient in the Gadash farm soil, while the Koc was similar in all soils except the Eden farm soil, in which it was more than twofold lower. In greenhouse studies, control of P. aegyptiaca was achieved at >2.5 ppb imazapic, but adequate control requires repeated applications due to the 7-day half-life (t1/2) of the herbicide in the soil. Tracking of imazapic in soil and tomato roots revealed that the herbicide accumulates in the tomato host plant roots, but its movement to newly formed roots is limited. The data obtained in the laboratory and greenhouse studies provide invaluable knowledge for devising field imazapic application strategies via drip irrigation systems for efficient and selective broomrape control.

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.

Molecular Tracking and Remote Sensing to Evaluate New Chemical Treatments Against the Maize Late Wilt Disease Causal Agent, Magnaporthiopsis maydis.

Late wilt is a destructive disease of corn: outbreaks occur at the advanced growth stage and lead to severe dehydration of susceptible hybrids. The disease's causal agent is the fungus Magnaporthiopsis maydis, whose spread relies on infested soils, seeds, and several alternative hosts. The current study aimed at advancing our understanding of the nature of this plant disease and revealing new ways to monitor and control it. Two field experiments were conducted in a heavily infested area in northern Israel seeded with highly sensitive corn hybrid. The first experiment aimed at inspecting the Azoxystrobin (AS) fungicide applied by spraying during and after the land tillage. Unexpectedly, the disease symptoms in this field were minor and yields were high. Nevertheless, up to 100% presence of the pathogen within the plant's tissues was measured using the quantitative real-time PCR method. The highest AS concentration tested was the most effective treatment, and resulted in a 6% increase in cob yield and a 4% increase in A-class yield. In the second experiment conducted in the following summer of the same year in a nearby field, the disease outbreak was dramatically higher, with about 350 times higher levels of the pathogen DNA in the untreated plots' plants. In this field, fungicide mixtures were applied using a dripline assigned for two coupling rows. The most successful treatment was AS and the Difenoconazole mixture, in which the number of infected plants decreased by 79%, and a 116% increase in crop yield was observed, along with a 41% increase in crop quality. Evaluation of the effectiveness of the treatments on the plants' health using a remote, thermal infra-red sensitive camera supported the results and proved to be an essential research tool.

Evaluating Azoxystrobin Seed Coating Against Maize Late Wilt Disease Using a Sensitive qPCR-Based Method.

Harpophora maydis, a phytopathogenic fungus, causes late wilt, a severe vascular maize disease characterized by relatively rapid wilting of maize plants near fertilization. The disease is currently controlled using resistant varieties. Here, we evaluated seed coating efficiency with azoxystrobin against H. maydis in a series of in vitro and in vivo trials. A real-time polymerase chain reaction (qPCR)-based method was developed and proved to be a sensitive, accurate tool for monitoring H. maydis DNA inside infected seeds, sprouts, and tissues of mature plants. In the early growth stages, the chemical coating drastically reduced the pathogen DNA prevalence in host tissues and minimized the suppressing effect on the plants' biomass and development. In an infested field, the qPCR assay identified the pathogen 20 days after seeding, up to a month before conventional PCR detection. In the resistant fodder maize cultivar 32D99, which showed only minor disease symptoms, the seed coating blocked fungal progression and increased cob and plant weight by 39 and 60%, respectively. Nevertheless, this treatment was unable to protect a sensitive maize hybrid, cultivar Prelude, at the disease wilting breakout (60 days after sowing). These results encourage further examination of azoxystrobin and other fungicides in the field using the qPCR detection method to evaluate their efficiency.

Effective chemical protection against the maize late wilt causal agent, Harpophora maydis, in the field.

Late wilt, a disease severely affecting maize fields throughout Israel, is characterized by relatively rapid wilting of maize plants before tasseling and until shortly before maturity. The disease's causal agent is the fungus Harpophora maydis, a soil-borne and seed-borne pathogen, which is currently controlled using reduced sensitivity maize cultivars. In a former study, we showed that Azoxystrobin (AS) injected into a drip irrigation line assigned for each row can suppress H. maydis in the field and that AS seed coating can provide an additional layer of protection. In the present study, we examine a more cost-effective protective treatment using this fungicide with Difenoconazole mixture (AS+DC), or Fluazinam, or Fluopyram and Trifloxystrobin mixture, or Prothioconazole and Tebuconazole mixture in combined treatment of seed coating and a drip irrigation line for two coupling rows. A recently developed Real-Time PCR method revealed that protecting the plants using AS+DC seed coating alone managed to delay pathogen DNA spread in the maize tissues, in the early stages of the growth season (up to the age of 50 days from sowing), but was less effective in protecting the crops later. AS+DC seed coating combined with drip irrigation using AS+DC was the most successful treatment, and in the double-row cultivation, it reduced fungal DNA in the host tissues to near zero levels. This treatment minimized the development of wilt symptoms by 41% and recovered cob yield by a factor of 1.6 (to the level common in healthy fields). Moreover, the yield classified as A class (cob weight of more than 250 g) increased from 58% to 75% in this treatment. This successful treatment against H. maydis in Israel can now be applied in vast areas to protect sensitive maize cultivars against maize late wilt disease.

Water purification from organic pollutants by optimized micelle-clay systems.

Removal of anionic pollutants (imazaquin, sulfentrazone, sulfosulfuron) and neutral pollutants (alachlor, acetochlor, chlorotoluron, bromacil) from water by micelles preadsorbed on montmorillonite was studied. Micelles of octadecyltrimethylammonium and benzyldimethylhexadecylammonium (BDMHDA) were used. The micelle-clay systems (1% w/w) removed 87-99% of the pollutants from their water solutions containing 1-33 mg/L of herbicide. The nature of the headgroup of the organic cation, which forms the micelles, is critical. Desorption of imazaquin and acetochlor from 0.3% (w/w) suspension of BDMHDA-clay complex after 24 h was around 7% in the range of adsorbed amounts from 0.6 to 15.3 mg/g. These results indicate rather slow rates and small extents of release of pollutants from micelle-clay complexes. Column filters (25 cm) made of a mixture of quartz sand and BDMHDA micelle-clay complex at 100:1 w/w ratio removed at least 99% of above pollutants from initial solutions containing 10 mg/L; 99.5 and 97% of sulfosulfuron and alachlor were removed from their initial solutions containing 200 and 5 microg/L, respectively. These data indicate that micelle-clay complexes are very efficient for water purification from organic contaminants.

Organoclay formulations of acetochlor: effect of high salt concentration.

This study aimed to evaluate new methodology for designing ecologically acceptable formulations of acetochlor. Modification of montmorillonite with phenyltrimethylammonium chloride (PTMA) or benzyltrimethylammonium chloride (BTMA) and organoclay formulations of acetochlor were prepared in the presence of high concentrations of sodium chloride (150 g/L). Acetochlor concentration in the equilibrium solutions was determined by HPLC. Release of acetochlor in a water system was performed by a funnel experiment. Leaching of acetochlor in soil was determined by a bioassay using a column technique and Setaria viridis as a test plant. The adsorbed amounts of acetochlor on montmorillonite exchanged by PTMA or BTMA were increased as NaCl concentration increased in the equilibrium solution. Leaching of acetochlor from organoclay formulations was significantly inhibited to the top soil layer (0-5 cm) when the formulations were prepared at extreme NaCl concentration (100-150 g/L). These results are in accord with a funnel experiment that showed a reduction in acetochlor release from the montmorillonite-based formulations. The application of this method for herbicide formulation would produce ecologically acceptable herbicide formulations that can significantly minimize the risk to groundwater pollution.

Sulfentrazone adsorbed on micelle-montmorillonite complexes for slow release in soil.

Interactions of the herbicide sulfentrazone with the cationic surfactants octadecyltrimethylammonium (ODTMA), hexadecyltrimethylammonium (HDTMA), and benzyldimethylhexadecylammonium (BDMHDA) have been studied for the design of slow-release formulations based on sulfentrazone adsorbed on a micelle-montmorillonite complex. Adsorbed amounts of sulfentrazone on ODTMA- and BDMHDA-montmorillonite complexes were 99.2-99.8% of that added, and desorption of herbicide in water during 24 h was low. After 10 washings in funnels with soil, only 2.6% of herbicide was released from ODTMA-montmorillonite formulations versus 100% release from the commercial formulation. The strong binding of sulfentrazone to micelles was confirmed by pH and spectroscopic measurements and was explained by the formation of ionic pairs between cationic surfactant and anionic herbicide. The ODTMA-clay and commercial formulations of sulfentrazone yield almost complete and 40% growth inhibition of green foxtail, respectively, at 700 g of active ingredient/ha. Hence, the slow release from micelle-clay formulations of sulfentrazone promotes its biological activity and reduces environmental contamination.

Sulfosulfuron incorporated in micelles adsorbed on montmorillonite for slow release formulations.

Slow release formulations of the anionic herbicide sulfosulfuron (SFS) were prepared by incorporating it in micelles of an organic cation octadecyltrimethylammonium, which adsorb on the clay-mineral montmorillonite. The fraction of SFS adsorbed on the micelle-clay complex reached 98%, whereas for monomer-clay complexes, its adsorption was insignificant. Fluorescence studies showed surface contact between the micelles and the clay surface. The rate of SFS release from the micelle-clay formulations in aqueous suspensions was slow (<1%, 72 h). Spraying SFS formulations on a thin soil layer in a funnel, followed by irrigations (50 mm), resulted in complete elution of SFS from the commercial formulation (dispersible granular) versus 4% from the micelle-clay formulation. A plant bioassay in Rehovot soil showed that these respective formulations yielded 23 and 65% of shoot growth inhibition of foxtail. Consequently, the slow release micelle-clay formulations of SFS yield significantly reduced leaching and enhanced biological activity, thus providing environmental and agricultural advantages.

Slow-release formulations of sulfometuron incorporated in micelles adsorbed on montmorillonite.

The design and tests of slow-release formulations of sulfometuron (SFM), an anionic sulfonylurea herbicide, are described. The formulations are based on incorporation of the herbicide in octadecyltrimethylammonium (ODTMA) micelles, which adsorb on a clay mineral, montmorillonite. An optimization of herbicide/micelle clay ratios yielded high adsorption of SFM (95%), and at a 1% (w/w) water suspension only 0.5% of the adsorbed SFM was released at times varying from hours to 9 days. An analytical test in Seville soil showed that under excessive irrigation (400 mm) 100% of the commercial formulation leached, whereas the micelle-clay formulations showed only 50-65% elution. A plant bioassay in Rehovot soil showed that the commercial dispersible granule formulation (Oust, 75% ai sulfometuron methyl) yielded only 23% root elongation inhibition at the top 5 cm of the soil, whereas complete inhibition was achieved with the micelle-clay formulation. The detected concentration of SFM for the micelle-clay formulation at a depth of 15-20 cm was half of that detected for the commercial one, indicating a reduction in leaching when applying the micelle-clay formulation. A 10-fold reduction in the applied dose of SFM in the micelle-clay formulations resulted in good herbicidal activity of 60-87% inhibition. These characteristics make the new formulation promising from the environmental and economic points of view.