Predation efficiency of the green lacewings Chrysoperla agilis and C. mutata against aphids and mealybugs in sweet pepper.

PEROXIDASE ACTIVITY AS AN INDICATOR OF WATER STRESS IN SWEET PEPPER PLANTS Antonio Evaldo Klar1; Sidnei Osmar Jadoski2; Giusepina Pace Pereira Lima31Departamento de Engenharia Rural, Faculdade de Ciências Agronômicas, Universidade Estadual Paulista, Botucatu, SP, klar@fca.unesp.br.2Universidade Estadual do Centro Oeste, Centro de Ciências Agrárias e Ambientais, Guarapuava, Pr3Departamento de Bioquímica, Faculdade de Ciências Agronômicas, Universidade Estadual Paulista, Botucatu, SP 1 ABSTRACT The purpose of the study was to evaluate the physiological and biochemical behavior of sweet pepper (Capsicum annuum L.) plants under different soil water availability conditions and the efficiency of the peroxidase (EC. 1.11.1.7) activity as an indicator of water stress in plants. The experiment was carried out at the Faculdade de Ciências Agronômicas – UNESP, Botucatu, SP. Sweet pepper plants were grown for 230 days after transplanting of seedlings and arranged in a completely randomized experimental design with 4 treatments, two irrigation managements (50 and 1500 kPa) and two soil surface managements (presence or absence of black polyethylene covering), and six replications. Physiological activities, such as stomatal transpiration and resistance to water vapor diffusion, were evaluated as well as biochemical activities, such as peroxidase activity and total soluble protein in foliar tissues. It was observed that soil water availability may lead to physiological and biochemical alterations in plants. Successive water stress cycles may promote the development of characteristics responsible for improving plant tolerance to periods of low water availability. The peroxidase enzyme activity showed to be an efficient indicator of water stress in sweet pepper plants. KEYWORDS: enzymatic activity; soil moisture; vegetable physiology; soluble protein; Capsicum annuum L. KLAR, A. E.; JADOSKI, S. O.; LIMA, G. P. P. A PEROXIDASE COMO INDICADOR DE DÉFICITS HÍDRICOS EM PLANTAS DE PIMENTÃO. 2 RESUMO O objetivo do presente estudo foi avaliar o comportamento fisiológico e bioquímico do pimentão (Capsicum annuum L) sob diferentes condições de disponibilidade de água no solo, além da eficiência da peroxidase (EC 1.11.1.7) como indicador de déficits hídricos nas plantas. O experimento foi desenvolvido na Faculdade de Ciências Agronômicas – UNESP, Botucatu, SP, durante 230 dias após o transplante das plântulas em condições de ambiente protegido com quatro tratamentos: dois níveis de irrigação (-50 e – 1500 kPa como potenciais mínimos de água do solo) e dois manejos de cobertura do solo (com e sem cobertura de lonas de polietileno preto) com seis repetições. As atividades fisiológicas, como a resistência difusiva dos estômatos e a transpiração foram avaliadas, assim como a peroxidase e a proteina solúvel, nos tecidos foliares. Os resultados mostraram que os déficits hídricos alteraram os fatores fisiológicos estudados e promoveram respostas positivas para a tolerância à déficits hídricos. A peroxidase mostrou ser um eficiente indicador de estresse hídrico em plantas de pimentão. UNITERMOS: irrigação, cobertura morta, potencial de água no solo, proteina solúvel.

Study of biochemical and histopathological changes induced in the sweet pepper (Capsicum annuum L.) in response to Chilli leaf curl virus infection

Effect of Host Plant on the Functional Response of the Predator Podisus nigrispinus (Heteroptera: Pentatomidae)

In March of 2013, new symptoms were observed in more than seven million nursery-grown sweet pepper (Capsicum annuum) plants in El Ejido, Almería (southern Spain). Symptoms included wilting without yellowing of leaves and stunting of plants. Plant crowns exhibited necrosis that advanced through the main root along with slight root rot. Xylem was not affected above or below the crown. Symptoms were thought to be caused by the well-known pepper pathogen Phytophthora capsici. However, sporodochia of Fusarium oxysporum were observed on plant crowns. Symptomatic seedlings (n = 200) were sampled and analyzed. Tissue from roots and epidermal crowns were plated on PDA, PARP, and Komada media, as well as stem discs on PDA and Komada. No Phytophthora sp. were observed and F. oxyporum was exclusively isolated from all 200 samples, from roots and crowns, but not from xylem. Pathogenicity of 60 of these F. oxysporum isolates was studied by inoculation onto sweet pepper plants (cv. del Piquillo) at the 2-true-leaf stage. Twelve plants per isolate, grown on autoclaved vermiculite, were inoculated by drenching with 20 ml of a conidial suspension (1 × 105 CFU/ml) of each isolate per plant. Each suspension was obtained by blending one PDA petri dish fully covered with one isolate. Non-inoculated plants served as control. Plants were maintained for 30 days in a growth chamber with a 14-h photoperiod (1.6 ×·104 lux) and temperatures at 23 to 26°C. The assay was conducted twice. Symptoms described above were reproduced on crown and roots of the inoculated plants with no symptoms in stem discs. No symptoms were observed on controls after 48 days. Host specificity was tested for 13 isolates to tomato (Solanum lycopersicum) cv. San Pedro, eggplant (S. melongena) cv. Alegria, cucumber (Cucumis sativus) cv. Marketmore, watermelon (Citrullus lanatus) cv. Sugar Baby, and Chinese cabbage (Brassica campestris subsp. condensa) cv. Kasumi (4). These plants were inoculated as previously described for pathogenicity tests (12 plants per species, repeated twice). None of the plants exhibited the characteristic symptoms after 60 days. Five isolates of F. oxysporum f. sp. radicis-cucumerinum and four isolates of F. o. f. sp radicis-lycopersici were also inoculated without any symptoms in any of the inoculated sweet pepper plants. Morphological identity of all isolates corresponded to F. oxysporum. The fungi were identified following the morphological keys and methodology provided by (1) and (2). Three isolates from the 60 tested were selected for molecular identification. Molecular identification was performed by sequencing partial TEF-1α gene (3). Subsequent database searches by BLASTn indicated that the resulting sequence of 659-bp had 100% identity with the corresponding gene sequence of F. oxysporum. The sequences were identical for the three isolates and were deposited on the EMBL Sequence Database (HG916993, HG916994, and HG916995). Results suggest that the pathogenic ability of the isolates varies from a vascular Fusarium wilt. F. oxysporum f. sp. capsici is a reported pathogen to sweet pepper (5), but the symptoms we have found are closer to those manifested by the formae speciales that causes root and crown rot of other plants. Consistent with the convention stablished for similar diseases we propose the name F. oxysporum f. sp. radicis-capsici f. sp. nov.

Sweet pepper, Capsicum annum L. is commercially grown vegetable grown for its nutritional and economical value. Sucking pests like aphids, whiteflies, thrips, mites, etc. are the major insect pest in sweet pepper fields. Farmers deliberately have been using chemical pesticides in their crop to manage various insect pest and such practices of using hazardous chemicals are harmful to human health and the biodiversity. Similarly, farmers don't have an idea of planting trap and/or companion plants and its role in natural control of pest. Hence, a study was conducted to know the population dynamics, especially to assess the aphid (Myzus persicae, Sulzer) population and its potential natural enemies by planting rapeseed as companion crop. This field experiment was conducted in six different locations of Bharatpur-23, Chitwan from Nov 2019 to Jan 2020 where three plots of sweet pepper were planted with rapeseed as companion crop and other three plots solely having no floral source around. Aphid population was recorded at weekly interval and its major natural enemies; ladybird beetle and syrphid fly. Abnormal data were squared root transformed and analyzed by using paired sample t-test. The result showed that the aphid population in sweet pepper field with companion crop was significantly lower than in sweet pepper grown without floral source (control). Highly significant number of ladybird beetles and syrphid flies were recorded in sweet pepper with companion crop compared to control. The finding is helpful to develop an integrated management protocol of sweet pepper pests with the practice of following conservation biological control strategy.

Causes of defoliation and low survival rate of grafted sweet pepper plants

The golden mosaic (begomovirus) and the yellowing (crinivirus) diseases are among the main viral diseases occurring in solanaceous crops in Brazil. A survey of viruses associated with both diseases was conducted on cultivated solanaceous plants from 2013 to 2017 to study their diversity and distribution in the Southeast/Midwest regions of Brazil. Samples from potato, eggplant, sweet pepper and tomato plants were collected in fields of seven Brazilian states (Bahia, Espirito Santo, Goias, Minas Gerais, Parana, Rio de Janeiro and Sao Paulo) and in the Federal District. Total RNA/DNA was extracted and tested by RT-PCR/PCR to detect the crinivirus tomato chlorosis virus (ToCV) and begomoviruses, respectively. Representative amplicons were directly sequenced for virus identification. Out of 343 samples, 54 were positive for ToCV: 38.6% in potato, 0.9% in sweet pepper, and 20.9% in tomato. For begomovirus detection, 234 samples were positive. In potato and sweet pepper plants, only tomato severe rugose virus (ToSRV) was detected, while four begomoviruses were detected in tomato plants. ToSRV was detected in 80.1% of the tomato samples, and was the predominant begomovirus. These results indicate a low diversity of crinivirus and begomovirus species infecting cultivated solanaceous crops in Brazil during the survey period.

Drought is a harmful abiotic factor that clearly affects the growth and production of plants. The negative impacts of drought can be reduced by ascorbic acid (AsA) which is an important non-enzymatic antioxidant in plants. The purpose of the current investigation was to study the effects of different levels of drought and the external application of AsA on sweet pepper plants. The experiment had two factors, the first factor was drought: irrigation within the field capacity, moderate stress (irrigation within the 60% field capacity) and severe stress (irrigation within the 30% field capacity). The second factor was AsA: 0 mM sprayed with distilled water, 0.5 and 1 mM. The experiment had three replications. The results illustrated that drought stress reduced the fresh and dry weight of roots and shoots. It also reduced the relative water content (RWC), diameter and length of fruit, chlorophyll content and leaf area. Drought stress increased the activity of polyphenol oxidase and ascorbate peroxidase enzymes. It caused improvements in antioxidant properties, total phenolic content, ascorbate and electrical conductivity (EC) in the leaves of sweet pepper. Higher concentrations of AsA by foliar application significantly increased the values of all measured parameters except the EC. The interaction between ascorbic acid and drought stress showed that ascorbic acid significantly increased the shoot fresh weight, root dry weight, antioxidant properties, ascorbate, polyphenol oxidase and ascorbate peroxidase. However, it reduced the electrical conductivity. It is claimed that sweet pepper plants acquire tolerance to drought because AsA contributes to an increase in antioxidant properties.

BIOLOGICAL CONTROL OF APHIDS BY THE PREDATORY MIDGE APHIDOLETES APHIDIMYZA IN THE PRESENCE OF INTRAGUILD PREDATORY BUGS AND THRIPS

Insect herbivory activates plant defense mechanisms and releases a blend of herbivore-induced plant volatiles (HIPVs). These volatile compounds may be involved in plant-plant communication and induce defense response in undamaged plants. In this work, we investigated whether the exposure of sweet pepper plants to HIPVs [(Z)-3-hexenol, (Z)-3-hexenyl acetate, (Z)-3-hexenyl propanoate, (Z)-3-hexenyl butanoate, hexyl butanoate, methyl salicylate and methyl jasmonate] activates the sweet pepper immune defense system. For this, healthy sweet pepper plants were individually exposed to the each of the above mentioned HIPVs over 48 h. The expression of jasmonic acid and salicylic acid related genes was quantified. Here, we show that all the tested volatiles induced plant defenses by upregulating the jasmonic acid and salicylic acid signaling pathway. Additionally, the response of Frankliniella occidentalis, a key sweet pepper pest, and Orius laevigatus, the main natural enemy of F. occidentalis, to HIPV-exposed sweet pepper plants were studied in a Y-tube olfactometer. Only plants exposed to (Z)-3-hexenyl propanoate and methyl salicylate repelled F. occidentalis whereas O. laevigatus showed a strong preference to plants exposed to (Z)-3-hexenol, (Z)-3-hexenyl propanoate, (Z)-3-hexenyl butanoate, methyl salicylate and methyl jasmonate. Our results show that HIPVs act as elicitors to sweet pepper plant defenses by enhancing defensive signaling pathways. We anticipate our results to be a starting point for integrating HIPVs-based approaches in sweet pepper pest management systems which may provide a sustainable strategy to manage insect pests in horticultural plants.

The tomato chlorosis virus (ToCV), transmitted by whitefly species of the genera Bemisia and Trialeurodes in a semipersistent manner, causes significant losses in solanaceous crops including tomato (Solanum lycopersicum) and sweet pepper (Capsicum annuum). Worldwide reports of natural and experimental infection of sweet pepper plants with ToCV are contradictory, raising the question of whether the critical factor determining infection is related to the susceptibility of sweet pepper cultivars or the genetics of virus isolates. In this work, ToCV isolates obtained from different hosts and geographical origins were biologically and molecularly analysed, transmitted by B. tabaci MEAM1 and MED, and the reaction of different sweet pepper cultivars was evaluated under different environmental conditions. Brazilian ToCV isolates from tomato, potato (S. tuberosum), S. americanum, and Physalis angulata did not infect plants of five sweet pepper cultivars when transmitted by B. tabaci MEAM1. Temperatures did not affect the sweet pepper susceptibility to tomato‐ToCV isolates from São Paulo, Brazil, and Florida, USA. However, sweet pepper‐ToCV isolates from Spain and São Paulo, Brazil, were transmitted efficiently to sweet pepper plants by B. tabaci MEAM1 and MED. Although the results indicated that ToCV isolates from naturally infected sweet pepper plants seem to be better adapted to plants of C. annuum, phylogenetic analyses based on the complete nucleotide sequences of RNA1 and RNA2 as well as the p22 gene did not reveal significant nucleotide differences among them. Additional studies are needed to identify intrinsic characteristics of ToCV isolates that favour infection of sweet pepper plants.

We investigated alfalfa and stinging nettle from the point of view of their potential to supply natural enemies of pests for protecting greenhouse cultures. We carried out a three year long study based on sweepnetting. The most frequent predatory insect was Orius niger (Wolff) in alfalfa and nettle. This species among others has an important potential in the biological control of thrips. Among ladybugs, the most important species in alfalfa and nettle were Propylea quatuordecimpunctata (Linnaeus), Coccinella septempunctata Linnaeus and Hippodamia variegata (Goeze), which consume significant amounts of aphids. The most frequent spider family in alfalfa and nettle was Thomisidae and Philodromidae. The dynamics of their prey composition suggests that these taxa present a significant suppressive force on pests. The predator thrips Aeolothrips intermedius Bagnall that feeds on phytophagous thrips, mites and other soft-bodied arthropods was also significant in the arthropod assemblage of alfalfa. Our three-year...

Zoophytophagous predators of the family Miridae (Heteroptera), which feed both on plant and prey, often maintain a close relationship with certain host plants. In this study, we aimed to select a suitable mirid predatory bug for aphid control in sweet pepper. Four species were compared: Macrolophus pygmaeus (Rambur), Dicyphus errans (Wolff), Dicyphus tamaninii Wagner and Deraeocoris pallens (Reuter). They were assessed on their establishment on sweet pepper plants with and without supplemental food (eggs of the flour moth Ephestia kuehniella Zeller and decapsulated cysts of the brine shrimp Artemia franciscana Kellogg) and on their effects on aphids with releases before and after aphid infestations. None of the predator species was able to control an established population of aphids on sweet pepper plants; however, the predators M. pygmaeus and D. tamaninii could successfully reduce aphid populations when released prior to an artificially introduced aphid infestation. The best results were achieved with M. pygmaeus in combination with a weekly application of supplemental food. Hence, our results demonstrate that the order and level of plant colonization by mirid predators and aphids determines how successful biological control is. Further studies are needed to evaluate the performance of mirid predatory bugs in sweet pepper crops in commercial greenhouses with multiple pests and natural enemies, in particular to understand how increased variation in food sources affects their feeding behaviour and preferences.

In nature, plants continuously face the attack from insect herbivores. To defend themselves, plants have evolved a plethora of direct and indirect defence mechanisms that are present either constitutively or induced upon insect herbivory. In response to insect herbivory, plants activate phytohormonal signal-transduction pathways, transcriptional responses and biological processes. These changes in transcriptional and biological processes level influence the plant’s phenotype and consequently plant ecology during the current season or over seasons. To gain more insight into the dynamics of plant phenotype, elucidating transcriptomic responses to insect herbivory is critical. Various studies have investigated whole-genome transcriptional responses of plants against different insect herbivores, but only few have reported on responses to cell-content feeding thrips. Thrips (Thysanoptera) are minute cell-content feeding insects and are serious pests on many commercial and ornamental plants. Thrips species such as western flower thrips (WFT) and onion thrips are among the most devastating pests on e.g. sweet pepper and white cabbage plants, respectively. Therefore, using a high-density time-series approach, the focus of this thesis was to investigate whole-genome transcriptional responses of sweet pepper and white cabbage plants upon WFT and onion thrips feeding, respectively, and the underlying genetic mechanisms. In addition, I focussed on one particular gene family, the lipoxygenases and a gene in this family that is involved in thrips-induced crop resistance. Chapter 2 was focused on the identification of the lipoxygenase gene family in pepper (Capsicum annuum). Lipoxygenases (LOXs) are non-heme, iron-containing dioxygenases involved in several developmental and defence-related plant processes, such as seed germination, fruit ripening, tuber development and JA-regulated plant defences. Through multiple in-silico analysis, a total of eight LOX genes were identified in pepper classifying four LOXs (CaLOX1, CaLOX3, CaLOX4 and CaLOX5) as 9-LOXs and four (CaLOX2, CaLOX6, CaLOX7 and CaLOX8) as 13-LOXs. Chapter 3 further narrowed down the LOX gene-family to one lipoxygenase (CaLOX2) gene through in-silico analysis and functionally characterized its involvement in jasmonate-dependent induced defence against WFT. Overall, this chapter shows involvement of CaLOX2 in JA-mediated plant resistance to thrips feeding. Chapter 4 elucidates the dynamics of transcriptional reprogramming of sweet pepper in response to WFT feeding. Approximately 8.6% (2060 up and 1002 downregulated) of the pepper genes were differentially expressed that categorized into 23 clusters (16 upregulated and 7 downregulated), each possessing a unique temporal expression pattern. Upregulated gene clusters were overrepresented with defence-related biological processes, whereas downregulated gene clusters were overrepresented with developmental processes. The transcription factor families ERF, MYB, NAC, bHLH and WRKY emerged as pivotal regulators in response to WFT feeding. The data show a chronological order in the activation of hormonal (JA, ET) and secondary metabolite (phenylpropanoids, flavonoids and terpenoids) pathways. Eventually, the comparative analysis of the WFT-induced transcriptional responses of Arabidopsis and sweet pepper plants to WFT feeding shows a conservation in the induction of the JA-pathway in both plants, whereas the majority of transcriptional responses are plant-specific. Chapter 5 focused on 1) elucidating whole-genome transcriptional reprogramming of white cabbage plants in response to onion thrips feeding and 2) comparative transcriptomics to disentangle similarities and differences in transcriptional responses between WFT-induced Arabidopsis and sweet pepper as well as onion-thrips-induced white cabbage. Approximately 9.7 % of the white cabbage genes showed differential expression with 48 (32 upregulated and 16 downregulated) gene clusters with upregulated clusters associated with defence and downregulated clusters with development-related biological processes. Phytohormone-related processes (JA, ET and SA) and secondary metabolite (phenylpropanoids, flavonoids, green-leaf volatiles and indolic glucosinolates) biosynthesis genes were induced, whereas aliphatic glucosinolate biosynthetic genes were suppressed. Comparative analysis of the transcriptional responses of Arabidopsis and sweet pepper to WFT and of white cabbage to onion thrips showed 1) conservation of the JA biosynthesis and signalling pathways, 2) conservation of involvement of TF families, such as MYB, bHLH and WRKY in regulating responses, 3) that the majority of the transcriptional responses to thrips are system-specific, 4) that genes involved in indole glucosinolate biosynthesis are upregulated, whereas genes involved in aliphatic glucosinolate biosynthesis are downregulated in both brassicaceous plants Arabidopsis and white cabbage and 5) that the white-cabbage transcriptomic response to onion thrips is relatively rapid and complex compared to the WFT-induced Arabidopsis and sweet pepper transcriptomic responses. The data presented in this thesis contributes to understand the intensity and complexity of dynamic plant transcriptional responses to thrips feeding, and this is discussed in Chapter 6.

Developed in a protected cultivation experiment evaluated the effects of nitrogen as urea and of potassium as potassium chloride on the concentration and micronutrient content for sweet pepper. Culture was constantly irrigated and developed in dark red latisol for 34 weeks. The production of fruits was continuous, with a total of 16 crops during the period. N increased the growth of the sweet pepper plants and decreased Fe, Mn, Cu and B levels in leaves and stems, besides decreasing Fe and Mn levels in the fruits, mainly up to dose 26.6g m -2 . Nevertheless, concentration of B and Cu in the fruits increased in a significant way at harvest time, after 11.5 weeks of planting. Increase of potassium doses resulted in a smaller growth of the sweet pepper plants and in higher levels of Fe and Mn in the stems and leaves, Cu in the stem and B in the fruits. After 34 weeks of growth, pepper leaves and stems concentrated more Fe, Mn and Cu, while B was more concentrated in the leaves and fruits. Highest level of B was present in the fruits, 101.7% and 64.6% respectively, at harvest time, after 11.5 and 31.5 weeks after the planting of sweet pepper. Mean concentrations of micronutrientes (mg kg -1 ) in stem, leaf and fruits of sweet pepper were respectively: Fe=169.7; 243.9; 126.4; Cu = 4.0; 3.3; 2.2; Mn=186; 323; 44.2; B=0.12; 38.9; 32.3. Sweet pepper plants accumulated higher amounts of Fe and B and lower amounts of Mn and Cu. Fruits had the highest extracted proportion during 34 weeks. Contents of micronutrientes in the plant and the export by the sweet pepper fruits were (mg m -2 ) B=225.3 (98%); Fe = 927.3 (92%); Cu=15.8 (91%); Mn=84.6 (78%);Cu=15.8 (91%);