Endophytic Colletotrichum siamense for Biocontrol and Resistance Induction in Guarana Seedlings.

Paullinia cupana var. sorbilis, known in Brazil as guarana plant, is an important plant and a major traditional crop in the State of Amazonas. It is a native Brazilian species of great economic and social importance, particularly in the Amazon region. Anthracnose caused by Colletotrichum spp. is the main challenge for this crop. Therefore, the present study verified whether C. siamense, an endophytic fungus infected with a mycovirus, could protect the seedlings and reduce or eliminate the characteristic symptoms. Total proteins and enzymatic activities of pathogenesis-related proteins (PRPs), including peroxidase (POX), chitinase (CHI), and phenylalanine ammonia lyase (PAL), were quantified. Guarana seedlings of cultivar Maués were sprayed with a C. siamense conidia suspension (5.0 × 103 conidia/mL). After ten days, the seedlings were sprayed with a suspension of the phytopathogen's conidia (1.0 × 106 conidia/mL). One group of these seedlings received the fungicide indicated for this crop. The fungicide was applied twice with an interval of 15 days between applications. Negative control seedlings did not receive any treatment (except water and fertilization), and positive control seedlings were treated only with the phytopathogen. The experiment was conducted between December 2019 and February 2020 in a greenhouse. The treatments were applied at an average temperature of 25°C and 85% relative humidity. Leaflets were randomly collected from each treatment group at 0, 48, 72, and 96 hours after pathogen inoculation and analyzed for total protein and enzyme production (POX, PAL, and CHI). After 28 days, the percentage of leaf lesions on the seedlings was evaluated. C. siamense inoculation reduced lesions. There were differences in total proteins and PRPs at different timepoints after inoculation, except for CHI activity, among treatments. To the best of our knowledge, this is the first record of resistance induction in guarana plants.

Morphostructural data and phylogenetic relationships of a new cnidarian myxosporean infecting spleen of an economic and ecological important bryconid fish from Brazil.

A new cnidarian myxosporean infecting the spleen of an economic and ecological important bryconid fish (Salminus franciscanus) is described based on integrative taxonomic approach including morphological, ultrastructural, biological traits, geography, molecular data and phylogenetic analysis. In a total of thirty specimens examined, nineteen (63.3%) were infected by an undescribed parasite species belonging to the genus Myxobolus. Plasmodial development was asynchronous, with young development in the periphery and mature myxospores in the central area and without projections and microvilli in the plasmodial wall. Mature myxospores were ovoid in shape and measured 7.9 ± 0.2 µm (7.6-8.1 µm) in length and 5.4 ± 0.1 µm (5.0-5.6 µm) in width. The two polar capsules were equal in size, occupying a little more than half of the myxospore body, measuring 4.0 ± 0.2 µm (3.9-4.1 µm) in length and 1.7 ± 0.1 µm (1.5-1.8 µm) in width. The polar tubules coiled in six turns, perpendicular to the long axis of polar capsule. Phylogenetic analysis placed the new species within a clade containing nine myxobolid species from South American characiforms fish and appears as a close species of Myxobolus pantanalis. Nevertheless, the sequences of the new species and M. pantanalis have a large genetic divergence of 13.5% in their SSU rDNA. In light of the differences observed from the integrative taxonomy, we confidently considered that this isolate is a new species of cnidarian myxosporean, M. douradae n. sp., increasing the knowledge of diversity of this enigmatic group of cnidarians.

Taxonomy, phylogeny and host-parasite interaction of two novel Myxobolus species infecting Brycon orthotaenia from the São Francisco River, Brazil.

Two new Myxobolus species were described infecting Brycon orthotaenia from the São Francisco River, in the state of Minas Gerais, Brazil. From a total of 39 B. orthotaenia collected, two specimens (5.1%) exhibited infection of the ovary and 12 specimens (30.8%) displayed infection of the liver. The plasmodia of both Myxobolus species were white and spherical measuring around 1 mm in length. The plasmodium found in the ovary showed mature myxospores, which were oval shaped from the frontal view and measured 9.2-11.0 (9.8 ± 0.4) µm in length, 5.9-6.9 (6.5 ± 0.3) µm in width and 4.6-5 (4.9 ± 0.1) µm in diameter. The two polar capsules were the same size and measured 3.9-6.2 (4.7 ± 0.5) µm in length and 1.8-2.4 (2.1 ± 0.2) µm in width. The polar tubules had 9 coils. The plasmodium found in the liver showed mature myxospores which were ellipsoidal in shape from the frontal view and measured 10.0-11.4 (10.7 ± 0.5) µm in length, 7.3-8.6 (8.1 ± 0.4) µm in width and 5.3-7.0 (6.8 ± 0.4) µm in diameter. The two polar capsules were the same size and measured 4.2-5.4 (4.9 ± 0.3) µm in length and 1.9-2.9 (2.7 ± 0.3) µm in width. The polar tubules had 8 coils. Ultrastructural analysis revealed an asynchronous sporogenesis process, with young developmental myxospore stages more often found in the periphery of the plasmodium and mature myxospores in the centre of the plasmodium. The plasmodial wall was formed by a single membrane which was not surrounded by a layer of host tissue. A thick layer of fibrous material was found in the peripheral ectoplasm close to the plasmodial wall of the plasmodium found in the ovary. Phylogenetic analysis based on the small-subunit ribosomal DNA - ssrDNA sequences and using the closest myxozoan sequences to each one of the species studied here based on previous GenBank data and Henneguya/Myxobolus/Thelohanellus species parasitizing fish from South American, revealed that the new species are grouped in a subclade together with other Myxobolus species parasitizing bryconid hosts.

Morphology and 18S rDNA sequencing of Henneguya peruviensis n. sp. (Cnidaria: Myxosporea), a parasite of the Amazonian ornamental fish Hyphessobrycon loretoensis from Peru: A myxosporean dispersal approach.

Myxosporean are endoparasitic cnidarians of wide distribution and responsible for important economic losses in fisheries and aquaculture. A new myxosporean species, Henneguya peruviensis n. sp., is herein described as obtained from the gill filaments of Hyphessobrycon loretoensis caught in the Nanay River, Department of Loreto, Peru. The parasite was found in 37 of 45 (82.2%) examined H. loretoensis. The new species was characterized based on morphological features and 18S rDNA gene sequence data. The sequencing of the 18S rDNA gene from the spores of H. peruviensis n. sp. resulted in 1632 nucleotides and this sequence did not match any of the myxozoan available in the GenBank. Phylogenetic analysis showed that H. peruviensis n. sp. closed together with H. leporinicola. Nonetheless, the 18S rDNA sequences of H. peruviensis n. sp. and H. leporinicola have only 82% similarity. This is the first description and molecular study of a Myxozoa parasitizing fish of the genus Hyphessobrycon in the Amazon basin. Given the importance of the ornamental fish industry in translocation of aquatic organisms worldwide, the international movement of myxosporeans in infected fish is discussed in terms of disease outbreaks and the need for preventative action.

Endophytic fungal diversity in Theobroma cacao (cacao) and T. grandiflorum (cupuaçu) trees and their potential for growth promotion and biocontrol of black-pod disease.

The endophytic niches of plants are a rich source of microbes that can directly and indirectly promote plant protection, growth and development. The diversity of culturable endophytic fungi from stems and branches of Theobroma cacao (cacao) and Theobroma grandiflorum (cupuaçu) trees growing in the Amazon region of Brazil was assessed. The collection of fungal endophytic isolates obtained was applied in field experiments to evaluate their potential as biocontrol agents against Phytophthora palmivora, the causal agent of the black-pod rot disease of cacao, one of the most important pathogens in cocoa-producing regions worldwide. The isolated endophytic fungi from 60 traditional, farmer-planted, healthy cacao and 10 cupuaçu plants were cultured in PDA under conditions inducing sporulation. Isolates were classified based upon the morphological characteristics of their cultures and reproductive structures. Spore suspensions from a total of 103 isolates that could be classified at least up to genus level were tested against P. palmivora in pods attached to cacao trees in the field. Results indicated that ∼70% of isolates showed biocontrol effects to a certain extent, suggesting that culturable endophytic fungal biodiversity in this system is of a mostly mutualistic type of interaction with the host. Eight isolates from genera Trichoderma (reference isolate), Pestalotiopsis, Curvularia, Tolypocladium and Fusarium showed the highest level of activity against the pathogen, and were further characterized. All demonstrated their endophytic nature by colonizing axenic cacao plantlets, and confirmed their biocontrol activity on attached pods trials by showing significant decrease in disease severity in relation to the positive control. None, however, showed detectable growth-promotion effects. Aspects related to endophytic biodiversity and host-pathogen-endophyte interactions in the environment of this study were discussed on the context of developing sustainable strategies for biological control of black-pod rot of cacao.

Genetic diversity in natural populations of Jacaranda decurrens Cham. determined using RAPD and AFLP markers.

Jacaranda decurrens (Bignoniaceae) is an endemic species of the Cerrado with validated antitumoral activity. The genetic diversity of six populations of J. decurrens located in the State of São Paulo was determined in this study by using molecular markers for randomly amplified polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP). Following optimization of the amplification reaction, 10 selected primers generated 78 reproducible RAPD fragments that were mostly (69.2%) polymorphic. Two hundred and five reproducible AFLP fragments were generated by using four selected primer combinations; 46.3% of these fragments were polymorphic, indicating a considerable level of genetic diversity. Analysis of molecular variance (AMOVA) using these two groups of markers indicated that variability was strongly structured amongst populations. The unweighted pair group method with arithmatic mean (UPGMA) and Pearson's correlation coefficient (RAPD -0.16, p = 0.2082; AFLP 0.37, p = 0.1006) between genetic matrices and geographic distances suggested that the population structure followed an island model in which a single population of infinite size gave rise to the current populations of J. decurrens, independently of their spatial position. The results of this study indicate that RAPD and AFLP markers were similarly efficient in measuring the genetic variability amongst natural populations of J. decurrens. These data may be useful for developing strategies for the preservation of this medicinal species in the Cerrado.

Genetic diversity in natural populations of Jacaranda decurrens Cham. determined using RAPD and AFLP markers

Jacaranda decurrens (Bignoniaceae) is an endemic species of the Cerrado with validated antitumoral activity. The genetic diversity of six populations of J. decurrens located in the State of São Paulo was determined in this study by using molecular markers for randomly amplified polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP). Following optimization of the amplification reaction, 10 selected primers generated 78 reproducible RAPD fragments that were mostly (69.2 percent) polymorphic. Two hundred and five reproducible AFLP fragments were generated by using four selected primer combinations; 46.3 percent of these fragments were polymorphic, indicating a considerable level of genetic diversity. Analysis of molecular variance (AMOVA) using these two groups of markers indicated that variability was strongly structured amongst populations. The unweighted pair group method with arithmatic mean (UPGMA) and Pearson's correlation coefficient (RAPD -0.16, p = 0.2082; AFLP 0.37, p = 0.1006) between genetic matrices and geographic distances suggested that the population structure followed an island model in which a single population of infinite size gave rise to the current populations of J. decurrens, independently of their spatial position. The results of this study indicate that RAPD and AFLP markers were similarly efficient in measuring the genetic variability amongst natural populations of J. decurrens. These data may be useful for developing strategies for the preservation of this medicinal species in the Cerrado.

Trichoderma martiale sp. nov., a new endophyte from sapwood of Theobroma cacao with a potential for biological control.

The new species Trichoderma martiale was isolated as an endophyte from sapwood in trunks of Theobroma cacao (cacao, Malvaceae) in Brazil. Based on sequences of translation-elongation factor 1-alpha (tef1) and RNA polymerase II subunit (rpb2) T. martiale is a close relative of, and morphologically similar to, T. viride, but differs in the production of discrete pustules on corn meal-dextrose agar (CMD) and SNA, in having a faster rate of growth, and in being a tropical endophyte. This new species was shown, in small-scale, in situ field assays, to limit black pod rot of cacao caused by Phytophthora palmivora, the cause of black pod disease.

Koninginins, phospholipase A2 inhibitors from endophytic fungus Trichoderma koningii.

Many isolated compounds from endophytic fungus have been useful to human beings, mainly those with medicinal applications and particularly those that can be used in inflammatory processes. Trichoderma fungi produce substances known as koninginins that have great structural similarity to compounds like flavonoids and vitamin E, which are able to inhibit the phospholipase A(2) (PLA(2)). In this work, koninginins A, E and F (KonA, KonE and KonF, respectivamente) isolated from Trichoderma koningii had their capabilities of inhibiting edema-inducing, myotoxic and enzymatic activities of the total venom of Bothrops jararacussu (jararacuçu) snake analyzed, as well as one of its homolog forms of phospholipases A(2) (bjPLA(2)-group IIB) and human secreted PLA(2) protein fusion (hsPLA(2)-group IIA). KonA was not efficient in inhibiting the three activities analyzed in all the tests performed. Nevertheless, KonE and KonF present great capability in inhibiting the effects provoked not only by the venom but also by both PLA(2). The activities inhibition shown by KonE and KonF over the enzymes is significantly higher than those obtained over the total venom. KonE and KonF were slightly more efficient in the inhibition of the group IIB (bjPLA(2)) PLA(2) effects than in the inhibition of the group IIA (hsPLA(2)) PLA(2) effects. KonE and KonF structures are similar to vitamin E and, possibly, the action mode of these molecules is similar to the one produced by the vitamin. These results, apparently, indicate that koninginins E and F, as well as vitamin E, present structural regions that might be used as start points in seeking for new and specific anti-inflammatory drugs against such enzymes.

Guarana (Paullinia cupana var. sorbilis), an anciently consumed stimulant from the Amazon rain forest: the seeded-fruit transcriptome.

Guarana (Paullinia cupana var. sorbilis) is a plant native to the central Amazon basin. Roasted seed extracts have been used as medicinal beverages since pre-Colombian times, due to their reputation as stimulants, aphrodisiacs, tonics, as well as protectors of the gastrointestinal tract. Guarana plants are commercially cultivated exclusively in Brazil to supply the national carbonated soft-drink industry and natural product stores around the world. In this report, we describe and discuss the annotation of 15,387 ESTs from guarana seeded-fruits, highlighting sequences from the flavonoid and purine alkaloid pathways, and those related to biotic stress avoidance. This is the largest set of sequences registered for the Sapindaceae family.

Bioprospecting highly diverse endophytic Pestalotiopsis spp. with antibacterial properties from Maytenus ilicifolia, a medicinal plant from Brazil.

Thirteen endophytic fungal strains of the genus Pestalotiopsis were isolated from the medicinal plant Maytenus ilicifolia Mart. ex. Reiss (commonly known as "espinheira santa") and their antimicrobial properties were investigated. Two isolates were successful in inhibiting the growth of the tested microorganisms (Escherichia coli, Klebsiella pneumoniae, Micrococcus luteus, Staphylococcus aureus, and methicillin-resistant Staphylococcus aureus (MRSA)) using the technique of bioautographic thin-layer chromatography (TLC) agar overlay assay. An analysis based on a polyphasic approach integrating taxonomic information, morphological traits, RAPD markers, and the sequencing of the ITS1-5.8S-ITS2 of the rDNA led to the assignment of the isolates as belonging to the species Pestalotiopsis microspora, Pestalotiopsis vismiae, and Pestalotiopsis leucothoes. Therefore, the present study presents a new approach to the study of endophytic fungi of the genus Pestalotiopsis.

Cloning and expression of an acidic platelet aggregation inhibitor phospholipase A2 cDNA from Bothrops jararacussu venom gland.

The phospholipase A2 (PLA2, E.C. 3.1.1.4) superfamily is defined by enzymes that catalyze the hydrolysis of the sn-2 bond of phosphoglycerides. Most PLA2s from the venom of Bothrops species are basic proteins, which have been well characterized both structurally and functionally, however, little is known about acidic PLA2s from this venom. Nevertheless, it has been demonstrated that they are non-toxic, with high catalytic and hypotensive activities and show the ability to inhibit platelet aggregation. To further understand the function of these proteins, we have isolated a cDNA that encodes an acidic PLA2 from a cDNA library prepared from the poly(A)+ RNA of venom gland of Bothrops jararacussu. The full-length nucleotide sequence of 366 base pairs encodes a predicted gene product with 122 amino acid with theoretical isoelectric point and size of 5.28 and 13,685 kDa, respectively. This acidic PLA2 sequence was cloned into expression vector pET11a (+) and expressed as inclusion bodies in Escherichia coli BL21(DE3)pLysS. The N-terminal amino acid sequence of the 14 kDa recombinant protein was determined. The recombinant acidic PLA2 protein was submitted to refolding and to be purified by RP-HPLC chromatography. The structure and function of the recombinant protein was compared to that of the native protein by circular dichroism (CD), enzymatic activity, edema-inducing, and platelet aggregation inhibition activities.

Cloning and identification of a complete cDNA coding for a bactericidal and antitumoral acidic phospholipase A2 from Bothrops jararacussu venom.

In order to better understand the function of acidic phospholipases A2 (PLA2s) from snake venoms, expressed sequence tags (ESTs) that code for acidic PLA2s were isolated from a cDNA library prepared from the poly(A)+ RNA of venomous glands of Bothrops jararacussu. The complete nucleotide sequence (366 bp), named BOJU-III, encodes the BthA-I-PLA2 precursor, which includes a signal peptide and the mature protein with 16 and 122 amino acid residues, respectively. Multiple comparison of both the nucleotide and respective deduced amino acid sequence with EST and protein sequences from databases revealed that the full-length cDNA identified (BOJU III--AY145836) is related to an acidic PLA2 sharing similarity, within the range 55-81%, with acidic phospholipases from snake venoms. Moreover, phylogenetic analysis of amino acid sequences of acidic PLA2s from several pit viper genera showed close evolutionary relationships among acidic PLA2s from Bothrops, Crotalus, and Trimeresurus. The molecular modeling showed structural similarity with other dimeric class II PLA2s from snake venoms. The native protein BthA-I-PLA2, a nontoxic acidic PLA2 directly isolated from Bothrops jararacussu snake venom, was purified and submitted to various bioassays. BthA-I-PLA2 displayed high catalytic activity and induced Ca2+-dependent liposome disruption. Edema induced by this PLA2 was inhibited by indomethacin and dexamethasone, thus suggesting involvement of the cyclo-oxygenase pathway. BthA-I-PLA2 showed anticoagulant activity upon human plasma and inhibited phospholipid-dependent platelet aggregation induced by collagen or ADP. In addition, it displayed bactericidal activity against Escherichia coli and Staphylococcus aureus and antitumoral effect upon breast adrenocarcinoma as well as upon human leukemia T and Erlich ascitic tumor. Following chemical modification with p-bromophenacyl bromide, total loss of the enzymatic and pharmacological activities were observed. This is the first report on the isolation and identification of a cDNA encoding a complete acidic PLA2 from Bothrops venom, exhibiting bactericidal and antitumoral effects.

Analysis of Bothrops jararacussu venomous gland transcriptome focusing on structural and functional aspects: I--gene expression profile of highly expressed phospholipases A2.

Snake venom glands are a rich source of bioactive molecules such as peptides, proteins and enzymes that show important pharmacological activity leading to in local and systemic effects as pain, edema, bleeding and muscle necrosis. Most studies on pharmacologically active peptides and proteins from snake venoms have been concerned with isolation and structure elucidation through methods of classical biochemistry. As an attempt to examine the transcripts expressed in the venom gland of Bothrops jararacussu and to unveil the toxicological and pharmacological potential of its products at the molecular level, we generated 549 expressed sequence tags (ESTs) from a directional cDNA library. Sequences obtained from single-pass sequencing of randomly selected cDNA clones could be identified by similarities searches on existing databases, resulting in 197 sequences with significant similarity to phospholipase A(2) (PLA(2)), of which 83.2% were Lys49-PLA(2) homologs (BOJU-I), 0.1% were basic Asp49-PLA(2)s (BOJU-II) and 0.6% were acidic Asp49-PLA(2)s (BOJU-III). Adjoining this very abundant class of proteins we found 88 transcripts codifying for putative sequences of metalloproteases, which after clustering and assembling resulted in three full-length sequences: BOJUMET-I, BOJUMET-II and BOJUMET-III; as well as 25 transcripts related to C-type lectin like protein including a full-length cDNA of a putative galactose binding C-type lectin and a cluster of eight serine-proteases transcripts including a full-length cDNA of a putative serine protease. Among the full-length sequenced clones we identified a nerve growth factor (Bj-NGF) with 92% identity with a human NGF (NGHUBM) and an acidic phospholipase A(2) (BthA-I-PLA(2)) displaying 85-93% identity with other snake venom toxins. Genetic distance among PLA(2)s from Bothrops species were evaluated by phylogenetic analysis. Furthermore, analysis of full-length putative Lys49-PLA(2) through molecular modeling showed conserved structural domains, allowing the characterization of those proteins as group II PLA(2)s. The constructed cDNA library provides molecular clones harboring sequences that can be used to probe directly the genetic material from gland venom of other snake species. Expression of complete cDNAs or their modified derivatives will be useful for elucidation of the structure-function relationships of these toxins and peptides of biotechnological interest.

cDNA sequence and molecular modeling of a nerve growth factor from Bothrops jararacussu venomous gland.

The complete nucleotide sequence of a nerve growth factor precursor from Bothrops jararacussu snake (Bj-NGF) was determined by DNA sequencing of a clone from cDNA library prepared from the poly(A) + RNA of the venom gland of B. jararacussu. cDNA encoding Bj-NGF precursor contained 723 bp in length, which encoded a prepro-NGF molecule with 241 amino acid residues. The mature Bj-NGF molecule was composed of 118 amino acid residues with theoretical pI and molecular weight of 8.31 and 13,537, respectively. Its amino acid sequence showed 97%, 96%, 93%, 86%, 78%, 74%, 76%, 76% and 55% sequential similarities with NGFs from Crotalus durissus terrificus, Agkistrodon halys pallas, Daboia (Vipera) russelli russelli, Bungarus multicinctus, Naja sp., mouse, human, bovine and cat, respectively. Phylogenetic analyses based on the amino acid sequences of 15 NGFs separate the Elapidae family (Naja and Bungarus) from those Crotalidae snakes (Bothrops, Crotalus and Agkistrodon). The three-dimensional structure of mature Bj-NGF was modeled based on the crystal structure of the human NGF. The model reveals that the core of NGF, formed by a pair of beta-sheets, is highly conserved and the major mutations are both at the three beta-hairpin loops and at the reverse turn.