Greenhouse environments often promote bacterial and fungal infections in important crop plants. Exogenous application of chemical inducers could help reduce the severity of infection, or even prevent infection. Small molecules such as glycerol, azelaic acid and pipecolic acid have been implicated as being important signaling molecules during Systemic Acquired Resistance (SAR). To examine if these small molecules could be used to induce resistance in crop plants, exogenous treatment assays were developed in cucumber. Glycerol spray and azelaic acid infiltration induced modest resistance at locally treated leaves. Pipecolic acid soil treatment induced modest resistance in aerial tissue of cucumber plants, and strong resistance when plants were treated weekly. This knowledge may be useful in promoting the commercialization of SAR-associated compounds to protect important crop plants against disease. Plants possess multiple defense pathways that include an SA signaling component to initiate resistance to microbial pathogens. However, during Age-Related Resistance (ARR) in Arabidopsis, a number of studies support that SA acts as an anti-microbial and anti-biofilm agent against Pseudomonas syringae pv. tomato (Pst) in the plant intercellular space. Little is known about the role of Pst biofilm formation during infection of young plants or if other defense responses act to suppress bacterial biofilm formation. Therefore Pst biofilm formation and the effect of PAMP Triggered Immunity (PTI) on bacterial biofilm formation was examined. PTI was induced with flg22 in wild-type Col-0, fls2, bak1-3 (PTI mutants) and sid2-2 (SA biosynthesis mutant). In vivo bacterial biofilm-like aggregate formation was monitored using Pst DC3000 PDSK-GFPuv and epifluorescence microscopy. Pst aggregate occurrence and size were positively correlated with bacterial success in susceptible plants (wild-type Col-0, fls2, bak1-3, sid2-2), while fewer and smaller bacterial aggregates were observed in Col-0 undergoing PTI. To determine if the extracellular polysaccharide, alginate was a major contributor to biofilm formation, in vivo bacterial aggregate formation was monitored using alginate deficient Pst-GFP. Alginate deficient Pst-GFP and wild-type Pst grew to similar levels in wild-type plants suggesting that the ability to produce alginate was not necessary for Pst pathogenicity and success in planta. Fewer alginate-deficient Pst aggregates were observed compared to wild-type Pst in inoculated plants, suggesting that the ability to produce alginate was modestly important for aggregate formation. These data provide novel insights into how biofilms form in planta, the association between pathogen virulence and biofilm formation, and how plant defense responses such as PTI not only reduce bacterial growth, but also target biofilms. / Thesis / Master of Science (MSc)

Systemic acquired resistance (SAR) is a form of inducible defense response triggered upon localized infection that confers broad-spectrum disease resistance against secondary infections. Several factors are known to regulate SAR and these include phenolic phytohormone salicylic acid (SA), phosphorylated sugar glycerol-3-phosphate (G3P), and dicarboxylic acid azelaic acid (AzA). This study evaluated a role for free radicals nitric oxide (NO) and reactive oxygen species (ROS) in SAR. Normal accumulation of both NO and ROS was required for normal SAR and mutations preventing NO/ROS accumulation and/or biosynthesis compromised SAR. A role for NO and ROS was further established using pharmacological approaches. Notably, both NO and ROS conferred SAR in a concentration dependent manner. This was further established using genetic mutants that accumulated high levels of NO. NO/ROS acted upstream of G3P and in parallel to SA. Collectively, these results suggest that NO and ROS are essential components of the SAR pathway.

The biological control agent Pseudomonas chlororaphis PA23 is effective at protecting Brassica napus (canola) from the necrotrophic fungus Sclerotinia sclerotiorum via direct antagonism. Despite the growing importance of biocontrol bacteria in protecting crop plants from fungal pathogens, little is known about how the host plant responds to bacterial priming on the leaf surface and certainly nothing about global changes in gene activity in the presence and absence of S. sclerotiorum. PA23 priming of mature canola plants reduced the number of lesion-forming petals by 90%. Global RNA sequencing of canola tissue at the host-pathogen interface showed a 16-fold reduction in the number of genes uniquely upregulated in response to S. sclerotiorum when pretreated with PA23. Upstream defense-related gene patterns suggest MAMP-triggered immunity via surface receptors detecting PA23 flagellin and peptidoglycans. Although systemic acquired resistance (SAR) was induced in all treatment groups, a response centered around a glycerol-3-phosphate (G3P)-mediated pathway was exclusively observed in canola plants treated with PA23 alone. Activation of these defense mechanisms by PA23 involved production of reactive oxygen species as well as pronounced thylakoid membrane structures and plastoglobule formation in leaf chloroplasts. PA23 therefore primes defense responses in the plant through the induction of unique local and systemic regulatory networks. / October 2016

Salicylic acid binding protein 2 (SABP2) is known to play a key role in Salicylic acid mediated defense pathway. SBIP-470 is SABP2 interacting protein that might be putatively involved in transfer of lipids. SBIP-470 was cloned without the signal peptide and expressed in E. coli. In vitro lipid binding assay using recombinant SBIP-470 failed to detect lipid binding. In vitro lipid transfer assay showed recombinant SBIP-470 does not transfer phospholipid. Study has shown that SBIP-470 is highly inducible upon infection with viral as well as bacterial pathogens. Induction of SBIP-470 expression upon the TMV infection most likely depends upon the SABP2 while its expression upon non-host bacterial pathogens is most probably inhibited by the SABP2. A study of Arabidopsis knockout mutants (ltp12 mutant and ltp2 mutant) lacking the SBIP-470 homolog genes showed defects in growth phenotype, and they were found susceptible to bacterial pathogens.

Fungicide use with different mechanisms of action as well the pulverization moment are the factors to be considered on the asian soybean rust control. Since many products have shown lost of efficiency, emerges the need of new alternatives to the control of soybean rust. The study aimed to quantify the effect of the systemic resistance inducer Acibenzolar-S-Methyl and the combination of fungicides molecules of the triazole + strobilurin + carboxamide applied in different stages in relation to the soybean rust control and yield. Two field experiments were conducted in greenhouse on the harvest year of 2010/2011. The experiment 1, with the pathogen inoculation at the phenological stage R5.1, was performed on factorial scheme (2x5x6), and the factors consisted of: Factor A: two soybeans cultivars (BMX Potência RR and Coodetec 219 RR); Factor D: treatments with Acibenzolar-S-Methyl (12,5 g a.i.ha-1), Azoxystrobin + Cyproconazole (60 + 24 g a.i.ha-1), Acibenzolar-SMethyl + Azoxystrobin + Cyproconazole (12,5 + 60 + 24 g a.i.ha-1), Pyraclostrobin + Epoxiconazole + Fluxapyroxad (64,8 + 40 + 40 g a.i.ha-1) and a testimony trait in each cultivar; factor E: Application of the treatments at the stages V6, R1, R5.1, 10, 17 and 24 days after the artificial inoculation (DAI) of Phakopsora pachyrhizi. The experiment 2, with the pathogen inoculation at the stage R1 was performed in factorial scheme (2x5x5) differing from the experiment 1 only on the factor E: application of the treatments in R1, 5, 10, 17 and 24 days after the inoculation. Was evaluated the area under the disease progress curve and the number of days to the appearance of the first pustule in leaflets of three distinct ages, also the average weight of seeds per experimental unit. The treatments presented differentiated behavior when exposed to different combinations between the cultivars, application period and leaflets age. Generally the combinations of Acibenzolar-S-Methyl + Azoxystrobin + Cyproconazole and Pyraclostrobin + Epoxiconazole + Fluxapyroxad resulted in better disease control and higher soybeans yield at the earlier and late applications in relation to the pathogen inoculation moment, without differing of Azoxystrobin + Cyproconazole in the others application moments. / O uso de fungicidas com diferentes mecanismos de ação, assim como a época de aplicação dos mesmos, são fatores que devem ser levados em consideração para o controle da ferrugem asiática na soja. Uma vez que muitos produtos têm apresentado perda de eficiência, surge a necessidade de novas alternativas de controle dessa doença. Com este trabalho buscou-se quantificar o efeito do uso do indutor de resistência Acibenzolar-S-Metil e da mistura de moléculas fungicidas dos grupos triazol + estrobilurina + carboxamida aplicados em diferentes épocas do ciclo da cultura em relação ao controle da ferrugem asiática e produtividade da soja. Para isso dois experimentos foram executados em estufa plástica na safra agrícola 2010/2011. O experimento 1, com a inoculação do patógeno no estádio fenológico R5.1, foi realizado em arranjo fatorial (2x5x6), cujos fatores foram compostos por: fator A: duas cultivares de soja (BMX Potência RR e Coodetec 219 RR); fator D: tratamentos com Acibenzolar-S-Metil (12,5 g i.a.ha-1), Azoxistrobina + Ciproconazol (60 + 24 g i.a.ha-1), Acibenzolar-S-Metil + Azoxistrobina + Ciproconazol (12,5 + 60 + 24 g i.a.ha-1), Piraclostrobina + Epoxiconazol + Fluxapyroxad (64,8 + 40 + 40 g i.a.ha-1) e um tratamento testemunha em cada cultivar; fator E: aplicação dos tratamentos em V6, R1, R5.1, 10 , 17 e 24 dias após a inoculação (DAI) artificial de Phakopsora pachyrhizi. O experimento 2, com a inoculação do patógeno no estádio fenológico R1, foi realizado em arranjo fatorial (2x5x5) diferindo-se do experimento 1 apenas para o fator E: aplicação dos tratamentos em R1, 5, 10 , 17 e 24 dias após a inoculação (DAI). Avaliou-se a área abaixo da curva de progresso da doença (AACPD) e o número de dias para aparecimento da primeira pústula em trifólios de três idades distintas, além do peso de grãos por parcela. Os tratamentos apresentaram comportamento diferenciado quando expostos a diferentes combinações entre cultivares, épocas de aplicação e idade de trifólios. De modo geral as misturas de Acibenzolar-S-Metil + Azoxistrobina + Ciproconazol e Piraclostrobina + Epoxiconazol + Fluxapyroxad resultaram em melhor controle da doença e maior produtividade de soja nas aplicações mais precoces e tardias em relação ao momento da inoculação do patógeno, não diferindo de Azoxistrobina + Ciproconazol nas demais épocas de aplicação dos tratamentos.

Tobacco SABP2, a 29. kDa protein catalyzes the conversion of methyl salicylic acid (MeSA) into salicylic acid (SA) to induce SAR. Pretreatment of plants with acibenzolar-. S-methyl (ASM), a functional analog of salicylic acid induces systemic acquired resistance (SAR). Data presented in this paper suggest that SABP2 catalyzes the conversion of ASM into acibenzolar to induce SAR. Transgenic SABP2-silenced tobacco plants when treated with ASM, fail to express PR-1 proteins and do not induce robust SAR expression. When treated with acibenzolar, full SAR is induced in SABP2-silenced plants. These results show that functional SABP2 is required for ASM-mediated induction of resistance.

Soybean cyst nematode (SCN), a major pathogen of soybean worldwide, causes huge losses in soybean production. Various approaches including cloning of genes to combat this devastating disease help to better understand the cellular function and immune responses of plants. Membrane fusion genes are the important regulatory parts of vesicular transport system, which works through packaging of intracellular compounds and delivering them to apoplast or nematode feeding sites to induce an incompatible reaction. The incompatible nature of membrane fusion proteins such as SNAP25, Munc18, Syntaxin, Synaptobrevin, NSF, Synaptotagmin and alpha-SNAP are conserved in eukaryotes and regulate the intracellular function to combat abiotic and biotic stress in plants. Overexpression of these genes in G. max [Williams 82(PI518671)] which is a susceptible cultivar of soybean to nematodes resulted in a reduction of the SCN population providing further insights of molecular and genetic approaches to solve the SCN problems in agriculture.

Tobacco SABP2 has been shown to display high affinity for salicylic acid (SA) and methylsalicylate (MeSA) and plays an important role in SAR signal development. Using biochemical approach, SABP2 has been shown to demonstrate strong esterase activity in converting MeSA to SA. Recent study shows that tetra fluoroacetophenone, a synthetic analog of SA, competitively inhibits SABP2 esterase activity as well as suppresses SAR signal development in tobacco mosaic virus (TMV)-infected tobacco plants. Not much has been studied on the effect of pesticides on plant defenses. Because both AChE and SABP2 are esterase-like proteins belonging to α/β hydroxylase superfamily, we hypothesize that pesticides may inhibit the MeSA esterase activity of SABP2 and block SAR development. Biochemical and molecular biology techniques were used to test this hypothesis. SAR in tobacco-TMV plant-pathogen system is measured by significant decrease in TMV-induced lesion sizes in secondarily inoculated distal leaves.

Most rice protection methods have currently used toxic chemicals to control pathogens and pests, which leads to environmental pollution. Systemic acquired resistance (SAR) taking advantage of natural defence reaction of plants could be proposed as an alternative, ecologically friendly approach for plant protection. Its application into rice production could minimize the chemicals quantity used and could contribute to the decrease of environmental pollution and the development of sustainable agriculture. The research was conducted to select the most effective chemical and suitable method to improve the health of rice plants infected by grassy stunt disease in net-house of Can Tho University. SAR chemicals were used at very low concentrations (in mM). Results showed that the height of rice plants treated with SAR chemicals was higher than that of plants untreated. Besides, the number of diseased plants was reduced and the ratio of firm grain and yield increased when plants were applied by SAR. Among the used substances, oxalic acid provided the best systemic acquired resistance. With oxalic acid, seed soaking was better than seed coating in systemic acquired resistance against rice grassy stunt disease. / Hầu hết các phương pháp sản xuất lúa hiện nay đều sử dụng các hóa chất độc hại trong việc phòng trừ bệnh và côn trùng gây hại, nên dẫn đến ô nhiễm môi trường. Kích thích tính kháng lưu dẫn giúp kích hoạt cơ chế tự nhiên kháng bệnh của cây có thể là giải pháp bảo vệ thực vật thay thế an toàn với môi trường. Việc ứng dụng tiến bộ này vào trong sản xuất lúa có thể làm giảm lượng hóa chất sử dụng, đóng góp vào việc giảm thiểu ô nhiễm môi trường và sự phát triển của một nền nông nghiệp bền vững. Nghiên cứu đã được thực hiện tại nhà lưới trường Đại học Cần Thơ để tuyển chọn hóa chất và phương pháp sử dụng hóa chất để tăng cường sức khỏe giúp cây lúa vượt qua bệnh vàng lùn. Hóa chất kích kháng được sử dụng ở một nồng độ rất thấp (đơn vị là mM). Kết quả cho thấy chiều cao cây lúa khi xử lý chất kích kháng tốt hơn so đối chứng không xử lý. Bên cạnh đó, số cây lúa nhiễm bệnh giảm, tỉ lệ hạt chắc và năng suất tăng khi cây lúa được xử lý với chất kích kháng. Trong số các chất kích kháng đã sử dụng, acid oxalic cho hiệu quả vượt trội. Với chất acid oxalic, phương pháp ngâm hạt cho hiệu quả kích kháng tốt hơn phương pháp áo hạt.

Cultivars of Agrostis stolonifera showed weak and strong responsiveness to the systemic acquired resistance (SAR) activator, benzothiadiazole (BTH), or the induced systemic resistance (ISR) activator, 2R, 3R-butanediol (BD). Next Generation RNA sequencing was used to identify 2163 putative transcripts with increased expression in BTH versus water-treated A. stolonifera. Among three BTH-induced genes, AsASP-2 and AsHIR-1 were induced faster, while AsLOX-1 had stronger transient induction, in one out of two strongly BTH-responsive cultivars. Three ISR-responsive genes, AsGNS-5, AsOPR-4 and AsAOS-1, showed no greater induction or priming in the strongly versus weakly BD-responsive cultivars. Cultivars of A. stolonifera vary significantly in their response to defense activators, however this is not consistently related to defense gene expression. To quantify disease severity, chlorophyll fluorescence imaging of the maximum quantum efficiency of photosystem II (Fv/Fm) was tested on Nicotiana benthamiana infected with Colletotrichum orbiculare. Leaf areas of healthy, non-necrotic affected and necrotic tissue could be individually quantified, which demonstrated that BD delayed symptom development by approx. 24-hour and reduced non-necrotic affected tissue compared to controls. Chlorophyll fluorescence imaging can quantify and reveal novel features about induced disease resistance.