Immunity priming uncouples the growth-defense trade-off in tomato.

Plants have developed an array of mechanisms to protect themselves against pathogen invasion. The deployment of defense mechanisms is imperative for plant survival, but can come at the expense of plant growth, leading to the 'growth-defense trade-off' phenomenon. Following pathogen exposure, plants can develop resistance to further attack. This is known as induced resistance, or priming. Here, we investigated the growth-defense trade-off, examining how defense priming via systemic acquired resistance (SAR), or induced systemic resistance (ISR), affects tomato development and growth. We found that defense priming can promote, rather than inhibit, plant development, and that defense priming and growth trade-offs can be uncoupled. Cytokinin response was activated during induced resistance, and found to be required for the observed growth and disease resistance resulting from ISR activation. ISR was found to have a stronger effect than SAR on plant development. Our results suggest that growth promotion and induced resistance can be co-dependent, and that, in certain cases, defense priming can drive developmental processes and promote plant yield.

Development of Powdery Mildew Resistance in Cucumber Using CRISPR/Cas9-Mediated Mutagenesis of CsaMLO8.

Powdery mildew (PM) diseases may severely limit the production of various crops, including members of the family Cucurbitaceae. Successful PM infection relies on the Mildew Resistance Locus O (MLO) plant gene family, which encodes susceptibility factors essential for fungus penetration into the host cell. In cucumber (Cucumis sativus), natural mutations in CsaMLO8 confer resistance to the PM pathogen Podosphaera xanthii. Here, we used CRISPR/Cas9-mediated mutagenesis to generate PM resistance in the susceptible cucumber cultivar Ilan. Two transgene-free Csamlo8 CRISPR mutant lines (Csamlo-cr-1 and Csamlo-cr-2) were isolated, the first with a 5-bp deletion in exon 1, and the second harboring a 1,280-bp deletion and 10-bp insertion between exons 1 and 5. Both lines showed high resistance to PM under semicommercial growth conditions in the summer growing seasons of 2019 and 2021. These results provide the basis for generating transgene-free powdery mildew resistance in cucumber in any genetic background. This method can directly be employed on commercial cultivars and hybrid parental lines, and thereby substantially shorten and simplify the breeding process for PM resistance in cucumber.

Nutrient Elements Promote Disease Resistance in Tomato by Differentially Activating Immune Pathways.

Nutrient elements play essential roles in plant growth, development, and reproduction. Balanced nutrition is critical for plant health and the ability to withstand biotic stress. Treatment with essential elements has been shown to induce disease resistance in certain cases. Understanding the functional mechanisms underlying plant immune responses to nutritional elements has the potential to provide new insights into crop improvement. In the present study, we investigated the effect of various elements-potassium (K), calcium (Ca), magnesium (Mg), and sodium (Na)-in promoting resistance against the necrotrophic fungus Botrytis cinerea and the hemibiotrophic bacterium Xanthomonas euvesicatoria in tomato. We demonstrate that spray treatment of essential elements was sufficient to activate immune responses, inducing defense gene expression, cellular leakage, reactive oxygen species, and ethylene production. We report that different defense signaling pathways are required for induction of immunity in response to different elements. Our results suggest that genetic mechanisms that are modulated by nutrient elements can be exploited in agricultural practices to promote disease resistance.

Effect of Mineral Nutrition and Salt Spray on Cucumber Downy Mildew (Pseudoperonospora cubensis).

It was previously shown that spraying with CaCl2, MgCl2, KCl, and K2SO4 and high N and Mg concentrations in the irrigation water of potted cucumber plants reduced powdery mildew, while medium P and high K concentrations increased powdery mildew. In the present work, we tested the effect of irrigation with N, P, K, Ca, and Mg and spraying with salts on downy mildew (Pseudoperonospora cubensis) of cucumber (CDM). In potted plants, an increase in N concentration in the irrigation water resulted in a major increase in CDM severity, while an increase in K or Ca concentrations resulted in a gradual increase in CDM severity. An increase in P and Mg concentrations in the irrigation water resulted in a major CDM decrease. Spraying with Ca, Mg, and K salts with Cl and SO4 anions resulted in CDM suppression in most cases, and a negative correlation was obtained between the salt and anion molar concentrations and the CDM severity. Using NaCl sprays, both Na and Cl concentrations were negatively related to the CDM severity. MgCl2 (0.1 M Cl), K2SO4 (0.1 M SO4), MgCl2 + K2SO4, and monopotassium phosphate (MKP, 1%) sprayed under commercial-like (CL) conditions significantly reduced CDM by 36.6% to 62.6% in one disease cycle, while, in a second cycle, CDM was significantly reduced only by K2SO4 and MKP. In conclusion, fertigation with P and Mg, and salt spraying decreased CDM, while only spraying under CL resulted in CDM suppression.

The Entomopathogenic Fungi Metarhizium brunneum and Beauveria bassiana Promote Systemic Immunity and Confer Resistance to a Broad Range of Pests and Pathogens in Tomato.

Biocontrol agents can control pathogens by reenforcing systemic plant resistance through systemic acquired resistance (SAR) or induced systemic resistance (ISR). Trichoderma spp. can activate the plant immune system through ISR, priming molecular mechanisms of defense against pathogens. Entomopathogenic fungi (EPF) can infect a wide range of arthropod pests and play an important role in reducing pests' population. Here, we investigated the mechanisms by which EPF control plant diseases. We tested two well studied EPF, Metarhizium brunneum isolate Mb7 and Beauveria bassiana as the commercial product Velifer, for their ability to induce systemic immunity and disease resistance against several fungal and bacterial phytopathogens, and their ability to promote plant growth. We compared the activity of these EPF to an established biocontrol agent, Trichoderma harzianum T39, a known inducer of systemic plant immunity and broad disease resistance. The three fungal agents were effective against several fungal and bacterial plant pathogens and arthropod pests. Our results indicate that EPF induce systemic plant immunity and disease resistance by activating the plant host defense machinery, as evidenced by increases in reactive oxygen species production and defense gene expression, and that EPF promote plant growth. EPF should be considered as control means for Tuta absoluta. We demonstrate that, with some exceptions, biocontrol in tomato can be equally potent by the tested EPF and T. harzianum T39, against both insect pests and plant pathogens. Taken together, our findings suggest that EPF may find use in broad-spectrum pest and disease management and as plant growth promoting agents.

Nutrient Status of Cucumber Plants Affects Powdery Mildew (Podosphaera xanthii).

We examined the effects of applications of N, P, K, Mg, and Ca through an irrigation solution and spraying K, Ca, and Mg salts on cucumber powdery mildew (CPM, Podosphaera xanthii) in potted plants and under commercial-like conditions. Spraying CaCl2 and MgCl2, or KCl and K2SO4, decreased CPM. There were significant negative correlations between the anion-related molar concentrations of the salts and disease severity. Among the sprayed treatments, NaCl provided significantly less CPM control when applied at a low (0.05 M) concentration, as compared with CaCl2 and MgCl2. When sprayed applications of Mg and K salts were analyzed separately from the untreated control, the Cl- salts were found to be more effective than the SO4-2 salts. High N and Mg concentrations in the irrigation water delivered to young, fruit-less cucumber plants reduced CPM, whereas more CPM was observed when the irrigation solution contained a medium amount of P and a high amount of K. In contrast, mature, fruit-bearing plants had less severe CPM at higher N, lower P, and higher K levels. Spraying mature plants with monopotassium phosphate, polyhalite (K2Ca2Mg(SO4)4·2H2O), and the salts mentioned above over an entire growing season suppressed CPM. CPM severity was also reduced by spray applications of Ca, Mg, and KSO4-2 and Cl- salts. Spray applications provided better CPM control than fertigation treatments. Induced resistance is probably involved in the effects of nutrients on CPM.

Effects of Microelements on Downy Mildew (Peronospora belbahrii) of Sweet Basil.

We recently demonstrated that spraying or irrigating with Ca, Mg and K reduces the severity of sweet basil downy mildew (SBDM). Here, the effects of Mn, Zn, Cu and Fe on SBDM were tested in potted plants. The effects of Mn and Zn were also tested under semi-commercial and commercial-like field conditions. Spray applications of a mixture of EDTA-chelated microelements (i.e., Fe-EDTA, Mn-EDTA, Zn-EDTA, Cu-EDTA and Mo) reduces SBDM severity. The application of EDTA chelates of individual microelements (i.e., Fe-EDTA, Mn-EDTA and Zn-EDTA) significantly reduces SBDM in potted plants. Foliar applications of Mn-EDTA and Zn-EDTA are found to be effective under semi-commercial conditions and were, thus, further tested under commercial-like conditions. Under commercial-like conditions, foliar-applied Mn-EDTA and Zn-EDTA decreased SBDM severity by 46-71%. When applied through the irrigation solution, those two microelements reduce SBDM by more than 50%. Combining Mg with Mn-EDTA and Zn-EDTA in the irrigation solution does not provide any additional disease reduction. In the commercial-like field experiment, the microelement-mixture treatment, applied as a spray or via the irrigation solution, was combined with fungicides spray treatments. This combination provides synergistic disease control. The mode of action in this plant-pathogen system may involve features of altered host resistance.

Effects of Agronomic Practices on the Severity of Sweet Basil Downy Mildew (Peronospora belbahrii).

Downy mildew (caused by Peronospora belbahrii) is a severe disease of sweet basil (Ocimum basilicum) crops around the world. We examined cultural methods for reducing the severity of sweet basil downy mildew (SBDM) under commercial conditions in greenhouses and walk-in tunnels. The effects of the orientation of walk-in tunnels, air circulation in greenhouses, plant density, and soil mulch were tested. SBDM was less severe in the tunnels that were oriented north-south than in those oriented east-west, but the yields in both types of tunnels were similar. Increased air circulation reduced SBDM severity, but did not affect yield. Gray or transparent polyethylene mulch reduced SBDM severity and, in most cases, increased yield relative to bare soil/growth medium. Yellow polyethylene mulch provided a smaller amount of control. The combination of increased air circulation and yellow polyethylene mulch provided synergistic SBDM control, whereas no synergism was observed when we combined increased air circulation with the other two types of mulch. Planting at half the usual density reduced disease severity. The reduced plant density was associated with reduced yield in the greenhouses, but not in the tunnels. All of the tested methods provided an intermediate level of SBDM control that varied among the different experiments.

Root zone warming represses foliar diseases in tomato by inducing systemic immunity.

Plants employ systemic-induced resistance as part of their defence arsenal against pathogens. In recent years, the application of mild heating has been found to induce resistance against several pathogens. In the present study, we investigated the effect of root zone warming (RZW) in promoting tomato's resistance against the necrotrophic fungus Botrytis cinerea (Bc), the hemibiotrophic bacterium Xanthomonas campestris pv. vesicatoria (Xcv) and the biotrophic fungus Oidium neolycopersici (On). We demonstrate that RZW enhances tomato's resistance to Bc, On and Xcv through a process that is dependent on salicylic acid and ethylene. RZW induced tomato immunity, resulting in increased defence gene expression, reactive oxygen species (ROS) and ethylene output when plants were challenged, even in the absence of pathogens. Overall, the results provide novel insights into the underlying mechanisms of warming-induced immune responses against phytopathogens with different lifestyles in tomato.

Gene Editing of the Decoy Receptor LeEIX1 Increases Host Receptivity to Trichoderma Bio-Control.

Fungal and bacterial pathogens generate devastating diseases and cause significant tomato crop losses worldwide. Due to chemical pesticides harming the environment and human health, alternative disease control strategies, including microorganismal bio-control agents (BCAs), are increasingly sought-after in agriculture. Bio-control microorganisms such as Trichoderma spp. have been shown to activate induced systemic resistance (ISR) in the host. However, examples of highly active bio-control microorganisms in agricultural settings are still lacking, due primarily to inconsistency in bio-control efficacy, often leading to widespread disease prior to the required ISR induction in the host. As part of its plant colonization strategy, Trichoderma spp. can secrete various compounds and molecules, which can effect host priming/ISR. One of these molecules synthesized and secreted from several species of Trichoderma is the family 11 xylanase enzyme known as ethylene inducing xylanase, EIX. EIX acts as an ISR elicitor in specific plant species and varieties. The response to EIX in tobacco and tomato cultivars is controlled by a single dominant locus, termed LeEIX, which contains two receptors, LeEIX1 and LeEIX2, both belonging to a class of leucine-rich repeat cell-surface glycoproteins. Both receptors are able to bind EIX, however, while LeEIX2 mediates plant defense responses, LeEIX1 acts as a decoy receptor and attenuates EIX induced immune signaling of the LeEIX2 receptor. By mutating LeEIX1 using CRISPR/Cas9, here, we report an enhancement of receptivity to T. harzianum mediated ISR and disease bio-control in tomato.

Systemic resistance to gray mold induced in tomato by benzothiadiazole and Trichoderma harzianum T39.

Gray mold (Botrytis cinerea) is an important disease of tomato (Solanum lycopersicum). This study examined defense-related gene expression involved in the resistance to B. cinerea that is induced in tomato plants by benzothiadiazole and Trichoderma harzianum T39 soil drench. In whole plants, transcriptional changes related to salicylic acid and ethylene were induced by the application of a 0.01% benzothiadiazole solution, whereas changes related to jasmonic acid were induced by the application of a 0.4% T39 suspension. On detached leaves, soil treatment by T39 led to enhanced resistance to B. cinerea infection that was proportional to the concentration of the T39 suspension. By 5 days after pathogen inoculation, the plants that had received the 0.04% T39 drench exhibited 62% less severe disease than the untreated plants. The 0.4% T39 drench led to an 84% reduction in disease severity. Observations of B. cinerea infection in leaves harvested from plants grown in the treated soils revealed that drenching with a T39 suspension induces systemic resistance against B. cinerea and primes salicylic acid- and ethylene-related gene expression in a manner proportional to the concentration of the biocontrol agent. Benzothiadiazole treatment induced resistance to gray mold independently of salicylic acid and led to strong priming of two genes known to be involved in defense against B. cinerea, Pti5 and PI2.

Expression and functional analyses of the plastid lipid-associated protein CHRC suggest its role in chromoplastogenesis and stress.

Chromoplastogenesis during flower development and fruit ripening involves the dramatic overaccumulation of carotenoids sequestered into structures containing lipids and proteins called plastid lipid-associated proteins (PAPs). CHRC, a cucumber (Cucumis sativus) PAP, has been suggested to be transcriptionally activated in carotenoid-accumulating flowers by gibberellin (GA). Mybys, a MYB-like trans-activator identified here, may represent a chromoplastogenesis-related factor: Its expression is flower specific and parallels that of ChrC during flower development; moreover, as revealed by stable ectopic and transient-expression assays, it specifically trans-activates ChrC promoter in flowers accumulating carotenoids and flavonoids. A detailed dissection of ChrC promoter revealed a GA-responsive element, gacCTCcaa, the mutation of which abolished ChrC activation by GA. This cis-element is different from the GARE motif and is involved in ChrC activation probably via negative regulation, similar to other GA-responsive systems. The GA responsiveness and MYBYS floral activation of the ChrC promoter do not overlap with respect to cis-elements. To study the functionality of CHRC, which is activated in vegetative tissues similar to other PAPs by various biotic and abiotic stresses, we employed a tomato (Lycopersicon esculentum) plant system and generated RNAi-transgenic lines with suppressed LeCHRC. Transgenic flowers accumulated approximately 30% less carotenoids per unit protein than controls, indicating an interrelationship between PAPs and flower-specific carotenoid accumulation in chromoplasts. Moreover, the transgenic LeCHRC-suppressed plants were significantly more susceptible to Botrytis cinerea infection, suggesting CHRC's involvement in plant protection under stress conditions and supporting the general, evolutionarily preserved role of PAPs.