TcTI, a Kunitz-type trypsin inhibitor from cocoa associated with defense against pathogens

Protease inhibitors (PIs) are important biotechnological tools of interest in agriculture. Usually they are the first proteins to be activated in plant-induced resistance against pathogens. Therefore, the aim of this study was to characterize a Theobroma cacao trypsin inhibitor called TcTI. The ORF has 740 bp encoding a protein with 219 amino acids, molecular weight of approximately 23 kDa. rTcTI was expressed in the soluble fraction of Escherichia coli strain Rosetta [DE3]. The purified His-Tag rTcTI showed inhibitory activity against commercial porcine trypsin. The kinetic model demonstrated that rTcTI is a competitive inhibitor, with a Ki value of 4.08 × 10–7 mol L−1. The thermostability analysis of rTcTI showed that 100% inhibitory activity was retained up to 60 °C and that at 70–80 °C, inhibitory activity remained above 50%. Circular dichroism analysis indicated that the protein is rich in loop structures and β-conformations. Furthermore, in vivo assays against Helicoverpa armigera larvae were also performed with rTcTI in 0.1 mg mL−1 spray solutions on leaf surfaces, which reduced larval growth by 70% compared to the control treatment. Trials with cocoa plants infected with Mp showed a greater accumulation of TcTI in resistant varieties of T. cacao, so this regulation may be associated with different isoforms of TcTI. This inhibitor has biochemical characteristics suitable for biotechnological applications as well as in resistance studies of T. cacao and other crops.

Variability and Virulence Analysis of Aspergillus niger Isolates Causing Collar Rot of Groundnut

Background: Collar rot of groundnut (Arachis hypogaea L.) caused by Aspergillus niger is a significant constraint in groundnut cultivation and responsible for huge economic losses in India including Rajasthan. Methods: By surveying of eight major groundnut growing districts of Rajasthan, India, one representative Aspergillus niger isolate from each district was established (ANBK-01= Bikaner, ANCH-02= Churu, ANDA-03= Dausa, ANJP-04= Jaipur, ANJL-05= Jalore, ANJD-06= Jodhpur, ANNG-07= Nagaur and ANSK-08= Sikar) for studying variability in the pathogen as well as to know the response of groundnut varieties to the highly virulent isolate. The colony and spore characteristics were observed for cultural and morphological variability. For resistance response to the disease, ten varieties (M-13, RG-633-9, RG-382, Girnar-2, RG-604, RG-578, Gajraj 10, RG-510, RG-632-1 and RG-644) were evaluated in the field for two consecutive years against a highly virulent Aspergillus niger (ANJP-04) isolate. Result: Our investigations cleared that all the isolates were showed cultural and morphological variability such as shape, colour and size of colony and size of conidia, conidiophores and columella. Isolate (ANJP-04) collected from Khejroli village of Chomu tehsil in Jaipur district showed maximum mycelial growth, conidia diameter, length and diameter of conidiophores and length and diameter of columella, early sporulation and found most virulent as it produced higher disease incidence (54.43%). Ten released varieties of groundnut in the field conditions, revealed that none of the variety was found completely free from the disease whereas RG-644, M-13 and RG-510 were found resistant while RG-604, Girnar-2, Gajraj-10 and RG-632-1 were found moderately resistant and rest were found susceptible to highly susceptible to the disease. Conclusively, it can be finalized that famers may cultivate these resistant varieties in areas where collar rot is a severe constraint. The conclusion of this study can also be utilized to screen varieties/genotypes of groundnut against highly virulent isolate for sustainability of breeding material to the disease effectively.

Two genomic regions of a sodium azide induced rice mutant confer broad-spectrum and durable resistance to blast disease

Rice blast, one of the most destructive epidemic diseases, annually causes severe losses in grain yield worldwide. To manage blast disease, breeding resistant varieties is considered a more economic and environment-friendly strategy than chemical control. For breeding new resistant varieties, natural germplasms with broad-spectrum resistance are valuable resistant donors, but the number is limited. Therefore, artificially induced mutants are an important resource for identifying new broad-spectrum resistant (R) genes/loci. To pursue this approach, we focused on a broad-spectrum blast resistant rice mutant line SA0169, which was previously selected from a sodium azide induced mutation pool of TNG67, an elite japonica variety. We found that SA0169 was completely resistant against the 187 recently collected blast isolates and displayed durable resistance for almost 20 years. Linkage mapping and QTL-seq analysis indicated that a 1.16-Mb region on chromosome 6 (Pi169-6(t)) and a 2.37-Mb region on chromosome 11 (Pi169-11(t)) conferred the blast resistance in SA0169. Sequence analysis and genomic editing study revealed 2 and 7 candidate R genes in Pi169-6(t) and Pi169-11(t), respectively. With the assistance of mapping results, six blast and bacterial blight double resistant lines, which carried Pi169-6(t) and/or Pi169-11(t), were established. The complementation of Pi169-6(t) and Pi169-11(t), like SA0169, showed complete resistance to all tested isolates, suggesting that the combined effects of these two genomic regions largely confer the broad-spectrum resistance of SA0169. The sodium azide induced mutant SA0169 showed broad-spectrum and durable blast resistance. The broad resistance spectrum of SA0169 is contributed by the combined effects of two R regions, Pi169-6(t) and Pi169-11(t). Our study increases the understanding of the genetic basis of the broad-spectrum blast resistance induced by sodium azide mutagenesis, and lays a foundation for breeding new rice varieties with durable resistance against the blast pathogen.

Polyketide Derivatives in the Resistance of Gerbera hybrida to Powdery Mildew

Powdery mildew is a common disease affecting the commercial production of gerbera flowers (Gerbera hybrida, Asteraceae). Some varieties show a certain degree of resistance to it. Our objective was to identify biomarkers of resistance to powdery mildew using an 1H nuclear magnetic resonance spectroscopy and chemometrics approach in a complex, fully factorial experiment to suggest a target for selection and breeding. Resistant varieties were found to differ from those that were susceptible in the metabolites of the polyketide pathway, such as gerberin, parasorboside, and gerberinside. A new compound probably involved in resistance, 5-hydroxyhexanoic acid 3-O-β-D-glucoside, was described for the first time. A decision tree model was built to distinguish resistant varieties, with an accuracy of 57.7%, sensitivity of 72%, and specificity of 44.44% in an independent test. Our results suggest the mechanism of resistance to powdery mildew in gerbera and provide a potential tool for resistance screening in breeding programs.

Integrated Transcriptome and Metabolome Analysis Reveals Key Metabolites Involved in Camellia oleifera Defense against Anthracnose

Camellia oleifera (Ca. oleifera) is a woody tree species cultivated for the production of edible oil from its seed. The growth and yield of tea-oil trees are severely affected by anthracnose (caused by Colletotrichum gloeosporioides). In this study, the transcriptomic and metabolomic analyses were performed to detect the key transcripts and metabolites associated with differences in the susceptibility between anthracnose-resistant (ChangLin150) and susceptible (ChangLin102) varieties of Ca. oleifera. In total, 5001 differentially expressed genes (DEGs) were obtained, of which 479 DEGs were common between the susceptible and resistant varieties and further analyzed. KEGG enrichment analysis showed that these DEGs were significantly enriched in tyrosine metabolism, phenylpropanoid biosynthesis, flavonoid biosynthesis and isoquinoline alkaloid biosynthesis pathways. Furthermore, 68 differentially accumulated metabolites (DAMs) were detected, including flavonoids, such as epicatechin, phenethyl caffeate and procyanidin B2. Comparison of the DEGs and DAMs revealed that epicatechin, procyanidin B2 and arachidonic acid (peroxide free) are potentially important. The expression patterns of genes involved in flavonoid biosynthesis were confirmed by qRT-PCR. These results suggested that flavonoid biosynthesis might play an important role in the fight against anthracnose. This study provides valuable molecular information about the response of Ca. oleifera to Co. gloeosporioides infection and will aid the selection of resistant varieties using marker-assisted breeding.

The Potential of Fungi as Bio-Control Agents in Managing Colletotrichum Kahawae In Kenya

Colletotrichum kahawae is a causative fungal agent of Coffee Berry Disease (CBD) that negatively affects coffee production. Its control relies on use of resistant varieties and chemical control. Fungicides used in control of CBD are costly and pose a risk to environment and health. The study was aimed at assessing the possible antagonistic potential of fungal isolates against Colletotrichum kahawae in in vitro conditions. Five isolates were used; one isolate was collected from berries while the rest were collected from soil. Testing for antagonism against Colletorichum kahawae was done by co-culturing the isolates on Potato Dextrose Agar. The degree of antagonism was determined by measuring and comparing the radial growth of pathogen with the bio-agent against the control. Out of the five isolates tested against C. kahawae, three isolates Fusarium proliferetum, Penicillium and Fusarium ceraneasum significantly inhibited test pathogen growth at 60%, 55% and 45.45%, respectively. The other isolates, Fumigatus aspergillus and Chaetomium perithecia showed inhibition of growth at 40% and 18.18% respectively. It was concluded that there is potential of using fungi as bio control of the coffee berry disease.

Screening of blast resistance genes in rice breeding samples

Rice is one of the most widespread and cultivated crops in the world. It is necessary to increase the yield of crops or expand their sown areas to resolve a food security problem in Russia. Current impossibility of expanding rice cultivated areas in the Rostov region and the need to maintain and increase its yield require developing new disease-resistant varieties. Rice genotypes with multiple blast resistance genes avoid significant yield losses. Since pyramiding and selection of resistance genes in the same genotype through traditional selection methods are complicated, it is urgent to search for homozygous samples using marker-assisted selection methods. This study was aimed to identify Pi-1, Pi-2, Pi-33 and Pi-ta blast resistance genes in breeding rice samples by MAS-methods. The study used CTAB-method for DNA-isolation, PCR, electrophoresis on agarose and polyacrylamide gels. The resulting gels were stained in a solution of ethidium bromide and photographed in ultraviolet light. To control the presence of blast resistance genes the following parental cultivars were used: C104LAC for the Pi-1 and Pi-33 genes, C101-A-51 for the Pi-2 gene, IR36 for the Pi-ta gene; Novator and Boyarin as controls of non-functional alleles of all studied genes. The 446 selection samples of the seventh generation were analyzed. As a result of the research, 127 rice samples that combine 2 or 3 different blast resistance genes were identified. The Pi-2 and Pi-33 genes combination was identified in 43 samples (1128/1, 1149/3, 1171/2, 1177/3, 1177/4, 1186/4, et al.). Samples with three resistance genes are the most interesting for selection and further breeding. For developing new blast-resistant varieties, we recommend using rice samples with the following combinations of resistance genes Pi-1+Pi-2+Pi-33 (1197/1, 1226/2, 1271/1, 1272/2), Pi-1+Pi-2+Pi-ta (1197/4, 1304/2, 1304/3, 1482/3, 1482/4, 1486/1) and Pi-2+Pi-33+Pi-ta (1064/1, 1064/3, 1281/2, 1281/3, 1281/4, 1282/2, 1283/1, 1283/2, 1284/3).

Assessment of Traditional Methods of Controlling Striga on Cultivated Crops in Sudanian Agricultural Zone of Chad

The phytoparasite, Striga spp., is a major biotic constraint to cereals production in sub-Saharan Africa. This study was carried out at station of Bébédjia of Chadian Institute of Agronomic Research for the Development in Sudanian agricultural zone of Chad, on traditional methods of controlling Striga spp. This study helped to find out several methods of controlling Striga among which the most important is the agronomic method including organic and or mineral fertilization, associated crops, crop rotation and the use of false hosts and trap crops. Some farmers cultivated resistant varieties and early maturing varieties to control pest attack and some others used shea flour and herbicide treatment to reduce the effect of Striga on the farm. Most of these methods were applied by farmers from generation to generation, without a thorough study to understand their mechanism. Thus, the listed methods will be analysed by research in station to find out scientific explanations for each applied method. Then experiments will be carried out to evaluate the effectiveness of some methods previously identified by extension rural services in farmers’ area after improvement.

The effectiveness of clove oil, its fractions, and clove oil-based fumigant tablet formulations, against Tribolium castaneum (Herbst)

Indonesia has seen an increase and widespread reports of resistance among stored-product insect pests to phosphine. The use of Syzigium aromaticum (clove oil) as an alternative fumigant may be a useful strategy to control infestation by phosphine-resistant varieties of stored-product insects. The objective of this study was to examine the effectiveness of whole (unfractionated) clove oil as well as its component fractions as a fumigant and repellent against the red flour beetle (Tribolium castaneum (Herbst)), and to develop a simple fumigant formulation for this purpose. The experimental design used to test the effectiveness of clove oil and its fractionation was a completely randomized design (CRD). Meanwhile, testing the effectiveness of tablet formulations was carried out by factorial CRD. Fumigation test results gave LD50 and LD95 values of 0.234 and 1.142 ml/l respectively, for crude clove oil used in a fumigation chamber against T. castaneum. An n-hexane fraction of clove oil tested under the same conditions was more lethal, causing 95% mortality of T. castaneum at the dose of 0.801 ml/l during fumigation. Finally, tablets containing a set proportion of clove oil and naphthalene (1:1) reached LD91 against T. castaneum after 7 days’ fumigation.

The effect of the crown shape on qualitative indicators of picking maturity and keeping capacity of apple fruits in the conditions of Crimea

В современных условиях развития интенсивного садоводства актуальной проблемой является подбор менее затратных и трудоемких систем формирования крон с соблюдением всех агротехнических мероприятий для получения высоких урожаев хорошего качества. Цель исследований направлена на изучение влияния разных способов формирования кроны на показатели съемной зрелости и лежкость плодов. Работа выполнялась в отделении «Крымская опытная станция садоводства» ФГБУН «НБС-ННЦ РАН» по методикам полевых исследований с плодовыми культурами. Объектами исследований являлись четыре формы кроны на подвое ЕМ-IХ (4 х 1 м, 2500 дер./га) и плоды сортов зимнего срока созревания: Джалита, Бреберн, Ренет Симиренко. Проведенные исследования позволили выявить наиболее эффективные формы кроны и достаточно устойчивые сорта для закладки интенсивных садов с высокой плотностью посадки, дающие высококачественную товарную продукцию, как в саду, так и в период хранения. Изменчивость показателей съемной зрелости в плодах зависит от сорта, погодных условий выращивания и формы кроны. Установлено, что плоды сортов Джалита, Бреберн (французская ось) и Ренет Симиренко (стройное веретено, безлидерная уплощенная, трехлидерная) в период хранения не имеют физиологических заболеваний. Дегустационная оценка вкуса составляет - 4,5-5,0 баллов, а естественная убыль не превышает 5,1%. Установлено также, что при хранении плоды сорта Джалита (стройное веретено, трехлидерная крона) повреждаются до 20% разными плодовыми гнилями, а плоды сорта Бреберн (стройное веретено) - подкожной пятнистостью - до 30%. Отмечено, что в период хранения происходит снижение плотности мякоти плодов в 1,4-2,2 раза, а также изменение вкуса в зависимости от сорта и формы кроны. In modern conditions of intensive gardening development, the actual problem is the selection of less costly and labor-intensive systems of crown shaping in compliance with all agrotechnical actions in order to obtain heavy yield of good quality. The goal of the research is aimed at studying the effect of different methods of crown shaping on the indicators of picking maturity and keeping capacity of fruits. The work was carried out in the department "Crimean experimental gardening station" of the FSBSI NBS-NSC RAS using methods of field research with fruit crops. The objects of research were four crown shapes on the EM-IX rootstock (4 x 1m, 2500 trees/ha) and the fruits of winter ripening varieties ‘Dzhalita’, ‘Brebern’, ‘Renet Simirenko’. The studies provided allow us to identify the most effective crown shapes and sufficiently resistant varieties for establishing intensive gardens with a high density of planting, giving high-quality marketable products, both in the garden and during storage. The variability of indicators of fruit picking maturity depends on the variety, weather conditions of growing and the crown shape. It was established that fruits of varieties ‘Dzhalita’, ‘Brebern’ (French axis) and ‘Renet Simirenko’ (slender spindle, leaderless flattened, three-leader) did not have physiological diseases during storage. Tasting assessment is 4.5-5.0 points, and the natural loss does not exceed 5.1%. It is also established that fruits of the ‘Dzalita’ variety (slender spindle, three-leader crown) during storage are subject to damage by various fruit rot up to 20%, and fruits of the ‘Brebern’ variety (slender spindle) by subcutaneous spotting - up to 30%. It is noted that during the storage period there is a decrease in the density of fruit pulp by 1.4 - 2.2 times, and a change in flavor depending on the variety and crown shape.