Arbuscular mycorrhiza‐mediated resistance in tomato against Cladosporium fulvum‐induced mould disease

Root colonization with arbuscular mycorrhizal fungi (AMF) enhances plant resistance particularly against soil‐borne pathogenic fungi. In this study, mycorrhizal inoculation with Glomus mosseae (Gm) significantly alleviated tomato mould disease caused by the air‐borne fungal pathogen, Cladosporium fulvum (Cf). The disease index (DI) in local leaves (receiving pathogen inoculation) and systemic leaves (just above the local leaf without pathogen inoculation) was 36.4% and 11.7% in mycorrhizal plants, respectively, whereas DI was 59.6% and 36.4% in the corresponding leaves of AMF non‐inoculated plants, after 50 days of Gm inoculation, corresponding to 15 days after Cf inoculation by leaf infiltration. Foliar spray inoculation with Cf also revealed that AMF pre‐inoculated plants had a higher resistance against subsequent pathogen infection, where the DI was 41.3% in mycorrhizal plants vs. 64.4% in AMF non‐inoculated plants. AMF‐inoculated plants showed significantly higher fresh and dry weight than non‐inoculated plants under both control (without pathogen) and pathogen treatments. AMF‐inoculated plants exhibited significant increases in activities of superoxide dismutase and peroxidase, along with decreases in levels of H₂O₂ and malondialdehyde, compared with non‐inoculated plants after pathogen inoculation. AMF inoculation led to increases in total chlorophyll contents and net photosynthesis rate as compared with non‐inoculated plants under control and pathogen infection. Pathogen infection on AMF non‐inoculated plants led to decreases in chlorophyll fluorescence parameters. However, pathogen infection did not affect these parameters in mycorrhizal plants. Taken together, these results indicate that AMF colonization may play an important role in plant resistance against air‐borne pathogen infection by maintaining redox poise and photosynthetic activity.     

Enhanced resistance in Theobroma cacao against oomycete and fungal pathogens by secretion of phosphatidylinositol‐3‐phosphate‐binding proteins

The internalization of some oomycete and fungal pathogen effectors into host plant cells has been reported to be blocked by proteins that bind to the effectors' cell entry receptor, phosphatidylinositol‐3‐phosphate (PI3P). This finding suggested a novel strategy for disease control by engineering plants to secrete PI3P‐binding proteins. In this study, we tested this strategy using the chocolate tree Theobroma cacao. Transient expression and secretion of four different PI3P‐binding proteins in detached leaves of T. cacao greatly reduced infection by two oomycete pathogens, Phytophthora tropicalis and Phytophthora palmivora, which cause black pod disease. Lesion size and pathogen growth were reduced by up to 85%. Resistance was not conferred by proteins lacking a secretory leader, by proteins with mutations in their PI3P‐binding site, or by a secreted PI4P‐binding protein. Stably transformed, transgenic T. cacao plants expressing two different PI3P‐binding proteins showed substantially enhanced resistance to both P. tropicalis and P. palmivora, as well as to the fungal pathogen Colletotrichum theobromicola. These results demonstrate that secretion of PI3P‐binding proteins is an effective way to increase disease resistance in T. cacao, and potentially in other plants, against a broad spectrum of pathogens.     

Overexpression of AtPAD4 in transgenic Brachypodium distachyon enhances resistance to Puccinia brachypodii

• Brachypodium distachyon (L.) has recently emerged as a model for temperate grasses for investigating the molecular basis of plant–pathogen interactions. Phytoalexin deficient 4 (PAD4) plays a regulatory role in mediating expression of genes involved in plant defence.
• In this research, we generated transgenic B. distachyon plants constitutively overexpressing AtPAD4. Two transgenic B. distachyon lines were verified using PCR and GUS phenotype.
• Constitutive expression of AtPAD4 in B. distachyon enhanced resistance to Puccinia brachypodii. P. brachypodii generated less urediniospores on transgenic than on wild‐type plants. AtPAD4 overexpression enhanced salicylic acid (SA) levels in B. distachyon‐infected tissues. qRT‐PCR showed that expression of pathogenesis‐related 1 (PR1) and other defence‐related genes were up‐regulated in transformed B. distachyon following infection with P. brachypodii.
• Our results indicate that AtPAD4 overexpression in B. distachyon plants led to SA accumulation and induced PR gene expression that reduced the rate of colonisation by P. brachypodii.

     

The Lr34 adult plant rust resistance gene provides seedling resistance in durum wheat without senescence

The hexaploid wheat (Triticum aestivum) adult plant resistance gene, Lr34/Yr18/Sr57/Pm38/Ltn1, provides broad‐spectrum resistance to wheat leaf rust (Lr34), stripe rust (Yr18), stem rust (Sr57) and powdery mildew (Pm38) pathogens, and has remained effective in wheat crops for many decades. The partial resistance provided by this gene is only apparent in adult plants and not effective in field‐grown seedlings. Lr34 also causes leaf tip necrosis (Ltn1) in mature adult plant leaves when grown under field conditions. This D genome‐encoded bread wheat gene was transferred to tetraploid durum wheat (T. turgidum) cultivar Stewart by transformation. Transgenic durum lines were produced with elevated gene expression levels when compared with the endogenous hexaploid gene. Unlike nontransgenic hexaploid and durum control lines, these transgenic plants showed robust seedling resistance to pathogens causing wheat leaf rust, stripe rust and powdery mildew disease. The effectiveness of seedling resistance against each pathogen correlated with the level of transgene expression. No evidence of accelerated leaf necrosis or up‐regulation of senescence gene markers was apparent in these seedlings, suggesting senescence is not required for Lr34 resistance, although leaf tip necrosis occurred in mature plant flag leaves. Several abiotic stress‐response genes were up‐regulated in these seedlings in the absence of rust infection as previously observed in adult plant flag leaves of hexaploid wheat. Increasing day length significantly increased Lr34 seedling resistance. These data demonstrate that expression of a highly durable, broad‐spectrum adult plant resistance gene can be modified to provide seedling resistance in durum wheat.     

Activation of Systemic Resistance to Magnaporthe oryzae in Rice by Substances Produced by Fusarium solani Isolated from Soil

Of 70 micro‐organisms (fungi, bacteria and actinomycetes) isolated from soil using vegetable tissue baits, 16 produced substances in culture fluids capable of preventing the development of blast caused by Magnaporthe oryzae on rice leaves with little or no inhibitory effect on the conidial germination of the pathogen. Isolate KS‐F14, which secreted substances capable of activating resistance in untreated leaves, was selected and identified as Fusarium solani. The resistance‐inducing substances were effective at pH values ranging from 5 to 10 and were stable under high temperatures, maintaining approximately the same level of activity even after autoclaving for 20 min. After application, the activated resistance in rice leaves persisted for 14 days. The polar solvent extracts of freeze‐dried KS‐F14 secretions were effective in activating resistance against M. oryzae in rice plants. The non‐polar solvent extracts were also effective, albeit not as effective as the polar solvent extracts, indicating that although the majority of the secreted resistance‐inducing compounds are hydrophilic, some of the compounds are hydrophobic. Treating secretions with cation or anion exchange resins only partially reduced their resistance‐inducing ability, suggesting that the resistance‐inducing components include both charged and non‐charged compounds. The resistance‐inducing compounds produced by F. solani have the potential to be developed into a commercial product for the control of rice blast and possibly other plant diseases.     

Feeding behavior and performance of Nasonovia ribisnigri on grafts,detached leaves,and leaf disks of resistant and susceptible lettuce

Aphids are dependent on the phloem sap of plants as their only source of nutrients. Host‐plant resistance in lettuce, Lactuca sativa L. (Asteraceae), mediated by the Nr gene is used to control the lettuce aphid Nasonovia ribisnigri (Mosely) (Hemiptera: Aphididae). The resistance is located in the phloem; however, the exact mechanism of resistance is unknown. In this study, we investigated whether the resistance factor (or factors) is synthesized in the root or in the shoot. The feeding behavior and performance of avirulent N. ribisnigri were studied on grafts of resistant and susceptible lettuce. In addition, the persistence of resistance in excised lettuce tissue was measured, by studying the feeding behavior and performance of N. ribisnigri on detached leaves and leaf disks of resistant lettuce. It appears that the resistance factor encoded by the Nr gene is produced in the shoots: aphid feeding was reduced on resistant shoots grafted on susceptible roots, whereas aphids were able to feed on grafts of susceptible shoots on resistant roots. Partial loss of resistance was observed after detachment of leaves and excision of leaf disks from resistant plants. Aphids fed longer on excised resistant plant tissue compared with intact resistant plants; however, compared with excised plant tissue of the susceptible cultivar, the time spent on feeding was shorter, indicating resistance was not completely lost. Our findings caution against the use of excised leaf material for aphid resistance bioassays.     

What happens in the pith stays in the pith: tissue‐localized defense responses facilitate chemical niche differentiation between two spatially separated herbivores

Herbivore attack is known to elicit systemic defense responses that spread throughout the host plant and influence the performance of other herbivores. While these plant‐mediated indirect competitive interactions are well described, and the co‐existence of herbivores from different feeding guilds is common, the mechanisms of co‐existence are poorly understood. In both field and glasshouse experiments with a native tobacco, Nicotiana attenuata, we found no evidence of negative interactions when plants were simultaneously attacked by two spatially separated herbivores: a leaf chewer Manduca sexta and a stem borer Trichobaris mucorea. T. mucorea attack elicited jasmonic acid (JA) and jasmonoyl‐l ‐isoleucine bursts in the pith of attacked stems similar to those that occur in leaves when M. sexta attacks N. attenuata leaves. Pith chlorogenic acid (CGA) levels increased 1000‐fold to levels 6‐fold higher than leaf levels after T. mucorea attack; these increases in pith CGA levels, which did not occur in M. sexta‐attacked leaves, required JA signaling. With plants silenced in CGA biosynthesis (irHQT plants), CGA, as well as other caffeic acid conjugates, was demonstrated in both glasshouse and field experiments to function as a direct defense protecting piths against T. mucorea attack, but not against leaf chewers or sucking insects. T. mucorea attack does not systemically activate JA signaling in leaves, while M. sexta leaf‐attack transiently induces detectable but minor pith JA levels that are dwarfed by local responses. We conclude that tissue‐localized defense responses allow tissue‐specialized herbivores to share the same host and occupy different chemical defense niches in the same hostplant.     

Red‐Light‐Induced Resistance to Brown Spot Disease Caused by Bipolaris oryzae in Rice

In this study, the protective effect of red light against the brown spot disease caused by the fungus Bipolaris oryzae in rice was investigated. Lesion formation was significantly inhibited on detached leaves that were inoculated with B. oryzae and kept under red for 48 h, but it was not inhibited when the leaves were kept under natural light or in the dark. The protective effect was also observed in intact rice plants inoculated with B. oryzae; the plants survived under red light, but most of them were killed by infection under natural light or dark condition. Red light did not affect fungal infection in onion epidermis cells or heat‐shocked leaves of rice, and it did not affect cellulose digestion ability; this suggested that the protective effect is due to red‐light‐induced resistance. In addition, the degree of protection increased as the red light dosage increased, regardless of the order of the red light and natural light period, indicating that red‐light‐induced resistance is time dependent. Feeding of detached leaves with a tryptophan decarboxylase inhibitor, s‐α‐fluoromethyltryptophan (0.1 mm ), for 24 h inhibited the development of resistance in response to red light irradiation. Suppression of resistance was also observed in leaves treated with a phenylalanine ammonia‐lyase inhibitor, α‐aminooxy acetic acid (0.5 mm ). These results suggest that the tryptophan and phenylpropanoid pathways are involved in the red‐light‐induced resistance of rice to B. oryzae.     

Suppression of Rust and Brown Eye Spot Diseases on Coffee by Phosphites and By‐products of Coffee and Citrus Industries

The rust and brown eye spot, caused by Hemileia vastatrix and Cercospora coffeicola, respectively, are the most important fungal diseases on coffee in South America. Their management is mainly by chemical treatment, and there is no genetic resistance to brown eye spot known so far. Considering the need for developing alternative products for their control, the goal of this work was to evaluate the effects of phosphites and by‐products of coffee and citrus industries on rust and brown eye spot. Formulations of coffee and citrus industry by‐products, phosphites and their combination with fungicide were evaluated in field experiments, and their effect on fungal urediniospores and conidia was evaluated in vitro. In the field, treatments were applied individually or in combination and the in vitro assays were performed with manganese phosphite (Reforce Mn), potassium phosphite and citrus industry by‐product (Fortaleza), copper phosphite and coffee industry by‐product (Fitoforce Full), and fungicide. The severity and incidence of rust and brown eye spot on coffee leaves, yield, and leaf retention were evaluated in the field. Percentage of spore germination was evaluated in vitro for both fungi, whereas mycelial growth was evaluated for C. coffeicola only. The treatments Fortaleza, Reforce Mn and Fitoforce Full suppressed both diseases with a reduction in defoliation. In the year 2012, the plants treated with Reforce Mn and Reforce Mn + Fortaleza showed a yield increase of 72 and 88%, respectively, which was similar to the results shown by the fungicide treatment. In vitro inhibition of germination of H. vastatrix urediniospores and of C. coffeicola conidia was observed and suggests that the products exert some toxic effects to both fungi. Finally, the results observed indicate that the combined use of by‐products of plant‐processing industries and phosphites is an alternative and can be added efficiently to the management of coffee diseases.     

The rice/maize pathogen Cochliobolus spp. infect and reproduce on Arabidopsis revealing differences in defensive phytohormone function between monocots and dicots

The fungal genus Cochliobolus describes necrotrophic pathogens that give rise to significant losses on rice, wheat, and maize. Revealing plant mechanisms of non‐host resistance (NHR) against Cochliobolus will help to uncover strategies that can be exploited in engineered cereals. Therefore, we developed a heterogeneous pathosystem and studied the ability of Cochliobolus to infect dicotyledons. We report here that C. miyabeanus and C. heterostrophus infect Arabidopsis accessions and produce functional conidia, thereby demonstrating the ability to accept Brassica spp. as host plants. Some ecotypes exhibited a high susceptibility, whereas others hindered the necrotrophic disease progression of the Cochliobolus strains. Natural variation in NHR among the tested Arabidopsis accessions can advance the identification of genetic loci that prime the plant’s defence repertoire. We found that applied phytotoxin‐containing conidial fluid extracts of C. miyabeanus caused necrotic lesions on rice leaves but provoked only minor irritations on Arabidopsis. This result implies that C. miyabeanus phytotoxins are insufficiently adapted to promote dicot colonization, which corresponds to a retarded infection progression. Previous studies on rice demonstrated that ethylene (ET) promotes C. miyabeanus infection, whereas salicylic acid (SA) and jasmonic acid (JA) exert a minor function. However, in Arabidopsis, we revealed that the genetic disruption of the ET and JA signalling pathways compromises basal resistance against Cochliobolus, whereas SA biosynthesis mutants showed a reduced susceptibility. Our results refer to the synergistic action of ET/JA and indicate distinct defence systems between Arabidopsis and rice to confine Cochliobolus propagation. Moreover, this heterogeneous pathosystem may help to reveal mechanisms of NHR and associated defensive genes against Cochliobolus infection.     

Dissecting virulence function from recognition: cell death suppression in Nicotiana benthamiana by XopQ/HopQ1‐family effectors relies on EDS1‐dependent immunity

Many Gram‐negative plant pathogenic bacteria express effector proteins of the XopQ/HopQ1 family which are translocated into plant cells via the type III secretion system during infection. In Nicotiana benthamiana, recognition of XopQ/HopQ1 proteins induces an effector‐triggered immunity (ETI) reaction which is not associated with strong cell death but renders plants immune against Pseudomonas syringae and Xanthomonas campestris pv. vesicatoria strains. Additionally, XopQ suppresses cell death in N. benthamiana when transiently co‐expressed with cell death inducers. Here, we show that representative XopQ/HopQ1 proteins are recognized similarly, likely by a single resistance protein of the TIR‐NB‐LRR class. Extensive analysis of XopQ derivatives indicates the recognition of structural features. We performed Agrobacterium‐mediated protein expression experiments in wild‐type and EDS1‐deficient (eds1) N. benthamiana leaves, not recognizing XopQ/HopQ1. XopQ recognition limits multiplication of Agrobacterium and attenuates levels of transiently expressed proteins. Remarkably, XopQ fails to suppress cell death reactions induced by different effectors in eds1 plants. We conclude that XopQ‐mediated cell death suppression in N. benthamiana is due to the attenuation of Agrobacterium‐mediated protein expression rather than the cause of the genuine XopQ virulence activity. Thus, our study expands our understanding of XopQ recognition and function, and also challenges the commonly used co‐expression assays for elucidation of in planta effector activities, at least under conditions of ETI induction.     

Inducible expression of a fusion gene encoding two proteinase inhibitors leads to insect and pathogen resistance in transgenic rice

Plant proteinase inhibitors (PIs) are considered as candidates for increased insect resistance in transgenic plants. Insect adaptation to PI ingestion might, however, compromise the benefits received by transgenic expression of PIs. In this study, the maize proteinase inhibitor (MPI), an inhibitor of insect serine proteinases, and the potato carboxypeptidase inhibitor (PCI) were fused into a single open reading frame and introduced into rice plants. The two PIs were linked using either the processing site of the Bacillus thuringiensis Cry1B precursor protein or the 2A sequence from the foot‐and‐mouth disease virus (FMDV). Expression of each fusion gene was driven by the wound‐ and pathogen‐inducible mpi promoter. The mpi‐pci fusion gene was stably inherited for at least three generations with no penalty on plant phenotype. An important reduction in larval weight of Chilo suppressalis fed on mpi‐pci rice, compared with larvae fed on wild‐type plants, was observed. Expression of the mpi‐pci fusion gene confers resistance to C. suppressalis (striped stem borer), one of the most important insect pest of rice. The mpi‐pci expression systems described may represent a suitable strategy for insect pest control, better than strategies based on the use of single PI genes, by preventing insect adaptive responses. The rice plants expressing the mpi‐pci fusion gene also showed enhanced resistance to infection by the fungus Magnaporthe oryzae, the causal agent of the rice blast disease. Our results illustrate the usefulness of the inducible expression of the mpi‐pci fusion gene for dual resistance against insects and pathogens in rice plants.     

Sex‐specific responses of Asian citrus psyllid to volatiles of conspecific and host‐plant origin

Diaphorina citri Kuwayama (Hemiptera: Psyllidae) is the primary vector of Candidatus Liberibacter spp. bacteria that cause citrus greening, a disease of worldwide importance. Olfactometry was employed to test responses of D. citri to odours from intact citrus plants (Mexican lime, Citrus aurantifolia, sour orange, Citrus aurantium, Marsh grapefruit, Citrus paradisi and Valencia orange, Citrus sinensis), citrus plants previously infested with D. citri, and odours of conspecifics including nymphs, adult insects of same and opposite sex, and their products (honeydew), both alone and in combination. In contrast to other studies, psyllids of both sexes were attracted to volatiles of undamaged Mexican lime leaves, whereas undamaged grapefruit attracted only females, and leaves of Valencia and sour orange did not attract either sex. All four plant species attracted female psyllids when previously infested, but only Mexican lime and sour orange‐attracted males. Thus, Citrus species appear to vary in the production of both constituitive and induced volatiles that attract adult psyllids. Volatiles emitted by nymphs did not attract either sex, but psyllid honeydew was attractive to males, likely due to female pheromone residues. Males oriented to the odour of females, whereas the reverse was not true, and neither males nor females oriented to same‐sex volatiles. The addition of conspecific cues (adults, nymphs or honeydew) did not increase female attraction to previously infested leaves, but male response was increased by the presence of adults and honeydew, regardless of plant species. Thus, female psyllids appear to orient more strongly to volatiles of plant origin, whereas males respond more strongly to cues emanating from females and conspecific excretions. These results suggest that female psyllids drive the initial colonization of host plants, whereas males orient to females and infested plants. Identification of the specific volatiles involved may permit their use in monitoring and management of this pest.     

Plant‐mediated interactions between two herbivores differentially affect a subsequently arriving third herbivore in populations of wild cabbage

• Plants are part of biodiverse communities and frequently suffer from attack by multiple herbivorous insects. Plant responses to these herbivores are specific for insect feeding guilds: aphids and caterpillars induce different plant phenotypes. Moreover, plants respond differentially to single or dual herbivory, which may cascade into a chain of interactions in terms of resistance to other community members. Whether differential responses to single or dual herbivory have consequences for plant resistance to yet a third herbivore is unknown.
• We assessed the effects of single or dual herbivory by Brevicoryne brassicae aphids and/or Plutella xylostella caterpillars on resistance of plants from three natural populations of wild cabbage to feeding by caterpillars of Mamestra brassicae. We measured plant gene expression and phytohormone concentrations to illustrate mechanisms involved in induced responses.
• Performance of both B. brassicae and P. xylostella was reduced when feeding simultaneously with the other herbivore, compared to feeding alone. Gene expression and phytohormone concentrations in plants exposed to dual herbivory were different from those found in plants exposed to herbivory by either insect alone. Plants previously induced by both P. xylostella and B. brassicae negatively affected growth of the subsequently arriving M. brassicae. Furthermore, induced responses varied between wild cabbage populations.
• Feeding by multiple herbivores differentially activates plant defences, which has plant‐mediated negative consequences for a subsequently arriving herbivore. Plant population‐specific responses suggest that plant populations adapt to the specific communities of insect herbivores. Our study contributes to the understanding of plant defence plasticity in response to multiple insect attacks.

     

Potential of bioinsecticidal Bacillus thuringiensis inoculum to suppress gray mold in tomato based on induced systemic resistance

The potential of the active ingredient of a commercial bioinsecticide, XenTari^® (Bacillus thuringiensis [BT] serovar aizawai strain ABTS‐1857), to suppress gray mold in tomato plants was elucidated. First, a suspension of the active ingredient of XenTari^® and a liquid culture of the bacterial strain as BT inocula were sprayed onto detached leaves or drenched into pots of tomato seedlings, and then, propagules of the gray mold fungus, Botrytis cinerea, were inoculated onto the leaves. The gray mold disease was significantly suppressed when rhizospheres were drenched with either inoculum, but not when inocula were sprayed onto detached leaves of seedlings. Both BT inocula were verified not to directly inhibit the mycelial growth of B. cinerea based on in vitro culture plate assays. Additionally, real‐time RT‐PCR analysis verified that the active ingredient increased the expression levels of defence‐related genes, such as PR‐1(P6) and P4, in the leaves of tomato seedlings. These results suggest that the active ingredient has the potential to suppress gray mold disease in tomato, not through direct antagonistic interactions with B. cinerea, but rather through systemic activation of the plant defence system by increased expression of several defence‐related genes.