Sporulation and Dispersal of the Biological Control Agent Aspergillus flavus AF36 Under Field Conditions.

Aflatoxins are carcinogens produced by the fungi Aspergillus flavus and A. parasiticus that contaminate pistachio crops. International markets reject pistachio when aflatoxins exceed permitted maximum levels. Releasing the atoxigenic strain AF36 of A. flavus is the leading aflatoxin pre-harvest control method. The product AF36 Prevail, sorghum grains coated with AF36 propagules, has been used in California since 2017. However, a high percentage of grains of the Prevail fail to sporulate in orchards. Here, the effect of soil moisture on the percentage of AF36 product grains sporulating (SG) and the quantity of spores per grain using a sporulation index (SI) was determined. Under controlled conditions, SG was higher than 85% when soil moisture was 13% or more, and SI increased with increasing soil moisture from 8.4 to 21%. The highest AF36 sporulation occurred near the micro-sprinklers when the grains were not impacted by the irrigation water drops. Arthropod predation was responsible for lost product grains, which was more pronounced in non-tilled soil than in tilled soil. Dispersal of the AF36 spores decreased markedly with the height and distance from the inoculum source, following a pattern of diffusion equations. However, AF36 spores easily reached canopies of pistachios located 10 m from the inoculum source. Our results indicate that AF36 Prevail should be applied close to the irrigation line in the moist soil area but avoiding the areas where excess irrigation causes water accumulation. The biocontrol of aflatoxins in California's pistachio production areas was optimized by improving the field realization of the biological control agent.

Pistachio Male Inflorescences as an Alternative Substrate for the Application of Atoxigenic Strains of Aspergillus flavus.

Aflatoxins are carcinogens mainly produced by Aspergillus flavus and A. parasiticus in susceptible crops, including pistachio. The primary inoculum sources of these pathogens are plant debris in the orchard soils. In Californian fields, one approach to controlling aflatoxin contamination is based on releasing the atoxigenic strain of A. flavus AF36 in inoculated (coated) sorghum grains (AF36 Prevail). However, this control method can fail due to poor sporulation of the AF36 strain or sorghum grain losses due to predation. In 2008 and 2018, we showed that toxigenic and atoxigenic isolates of Aspergillus spp. frequently colonized fallen inflorescences of male pistachio trees. Under controlled conditions, strain AF36 profusely colonized pistachio male inflorescences when humidity was higher than 90%. However, there were significant differences between types of inflorescence (aerial > fallen). In 2016, we considerably (P = 0.015) increased the population of AF36 on the canopies of trees when fallen inflorescences were inoculated with AF36, compared with untreated trees. In 2017 and 2018, these differences were not detected (P > 0.05) due to cross-contamination of strain AF36 between seasons and neighboring plots. In any case, the density of AF36 spores on the canopy of the inflorescence-treated trees was similar (P > 0.05) to that on trees treated with the commercial product. Here, we present a new method for applying strain AF36 based on using a natural, abundant, and uniformly distributed substrate in pistachio fields, and we discuss how it can be improved. Furthermore, our results indicate that, in pistachio orchards where biocontrol practices are not conducted, eliminating this important source of toxigenic Aspergillus inoculum is recommended.

Resistance to Aspergillus flavus and Aspergillus parasiticus in Almond Advanced Selections and Cultivars and Its Interaction with the Aflatoxin Biocontrol Strategy.

Aflatoxin contamination of almond kernels, caused by Aspergillus flavus and A. parasiticus, is a severe concern for growers because of its high toxicity. In California, the global leader of almond production, aflatoxin can be managed by applying the biological control strain AF36 of A. flavus and selecting resistant cultivars. Here, we classified the almond genotypes by K-Means cluster analysis into three groups (susceptible [S], moderately susceptible [MS], or resistant [R]) based on aflatoxin content of inoculated kernels. The protective effects of the shell and seedcoat in preventing aflatoxin contamination were also examined. The presence of intact shells reduced aflatoxin contamination >100-fold. The seedcoat provided a layer of protection but not complete protection. In kernel inoculation assays, none of the studied almond genotypes showed a total resistance to the pathogen. However, nine traditional cultivars and four advanced selections were classified as R. Because these advanced selections contained germplasm derived from peach, we compared the kernel resistance of three peach cultivars to that shown by kernels of an R (Sonora) and an S (Carmel) almond cultivar and five pistachio cultivars. Overall, peach kernels were significantly more resistant to the pathogen than almond kernels, which were more resistant than pistachio kernels. Finally, we studied the combined effect of the cultivar resistance and the biocontrol strain AF36 in limiting aflatoxin contamination. For this, we coinoculated almond kernels of R Sonora and S Carmel with AF36 72 h before or 48 h after inoculating with an aflatoxin-producing strain of A. flavus. The percentage of aflatoxin reduction by AF36 strain was greater in kernels of Carmel (98%) than in those of Sonora (83%). Cultivar resistance also affected the kernel colonization by the biological control strain. AF36 strain limited aflatoxin contamination in almond kernels even when applied 48 h after the aflatoxin-producing strain. Our results show that biocontrol combined with the use of cultivars with resistance to aflatoxin contamination can result in a more robust protection strategy than the use of either practice in isolation.

Quantification of the Aflatoxin Biocontrol Strain Aspergillus flavus AF36 in Soil and in Nuts and Leaves of Pistachio by Real-Time PCR.

The species Aspergillus flavus and A. parasiticus are commonly found in the soils of nut-growing areas in California. Several isolates can produce aflatoxins that occasionally contaminate nut kernels, conditioning their sale. Strain AF36 of A. flavus, which does not produce aflatoxins, is registered as a biocontrol agent for use in almond, pistachio, and fig crops in California. After application in orchards, AF36 displaces aflatoxin-producing Aspergillus spp. and thus reduces aflatoxin contamination. Vegetative compatibility assays (VCAs) have traditionally been used to track AF36 in soils and crops where it has been applied. However, VCAs are labor intensive and time consuming. Here, we developed a quantitative real-time PCR (qPCR) protocol to quantify proportions of AF36 accurately and efficiently in different substrates. Specific primers to target AF36 and toxigenic strains of A. flavus and A. parasiticus were designed based on the sequence of aflC, a gene essential for aflatoxin biosynthesis. Standard curves were generated to calculate proportions of AF36 based on threshold cycle values. Verification assays using pure DNA and conidial suspension mixtures demonstrated a significant relationship by regression analysis between known and qPCR-measured AF36 proportions in DNA (R2 = 0.974; P < 0.001) and conidia mixtures (R2 = 0.950; P < 0.001). Tests conducted by qPCR in pistachio leaves, nuts, and soil samples demonstrated the usefulness of the qPCR method to precisely quantify proportions of AF36 in diverse substrates, ensuring important time and cost savings. The outputs of this study will serve to design better aflatoxin management strategies for pistachio and other crops.

Experimental and theoretical studies on the rearrangement of 2-oxoazepane α,α-amino acids into 2'-oxopiperidine ß(2,3,3) -amino acids: an example of intramolecular catalysis.

Enantiopure ß-amino acids represent interesting scaffolds for peptidomimetics, foldamers and bioactive compounds. However, the synthesis of highly substituted analogues is still a major challenge. Herein, we describe the spontaneous rearrangement of 4-carboxy-2-oxoazepane α,α-amino acids to lead to 2'-oxopiperidine-containing ß(2,3,3) -amino acids, upon basic or acid hydrolysis of the 2-oxoazepane α,α-amino acid ester. Under acidic conditions, a totally stereoselective synthetic route has been developed. The reordering process involved the spontaneous breakdown of an amide bond, which typically requires strong conditions, and the formation of a new bond leading to the six-membered heterocycle. A quantum mechanical study was carried out to obtain insight into the remarkable ease of this rearrangement, which occurs at room temperature, either in solution or upon storage of the 4-carboxylic acid substituted 2-oxoazepane derivatives. This theoretical study suggests that the rearrangement process occurs through a concerted mechanism, in which the energy of the transition states can be lowered by the participation of a catalytic water molecule. Interestingly, it also suggested a role for the carboxylic acid at position 4 of the 2-oxoazepane ring, which facilitates this rearrangement, participating directly in the intramolecular catalysis.

Divergent, stereoselective access to heterocyclic α,α-quaternary- and ß(2,3,3)-amino acid derivatives from a N-Pmp-protected Orn-derived ß-lactam.

A suitably protected Orn-derived (3S,4S)-ß-lactam was used as common intermediate in the synthesis of conformationally constrained (3S,4S)-2-oxoazepane α,α- and (2S,3S)-2-oxopiperidine-ß(2,3,3)-amino acid derivatives. Compared to alternative procedures using an N-p-methoxybenzyl group at the 2-azetidinone, the incorporation of a p-methoxyphenyl moiety is crucial for the excellent stereochemical outcomes in the preparation of these heterocyclic amino acids. Chemoselective 7- or 6-exo-trig cyclization was achieved through alternative sequences of Pmp-deprotection/Boc-activation, followed by inter- and intramolecular ß-lactam ring opening, respectively.

Exploring the Phe-Gly dipeptide-derived piperazinone scaffold in the search for antagonists of the thrombin receptor PAR1.

A series of Phe-Gly dipeptide-derived piperazinones containing an aromatic urea moiety and a basic amino acid has been synthesized and evaluated as inhibitors of human platelet aggregation induced by the PAR1 agonist SFLLRN and as cytotoxic agents in human cancer cells. The synthetic strategy involves coupling of a protected basic amino acid benzyl amide to 1,2- and 1,2,4-substituted-piperazinone derivatives, through a carbonylmethyl group at the N1-position, followed by formation of an aromatic urea at the exocyclic moiety linked at the C2 position of the piperazine ring and removal of protecting groups. None of the compounds showed activity in the biological evaluation.

Study of the competition between Colletotrichum godetiae and C. nymphaeae, two pathogenic species in olive.

Olive anthracnose, a critical olive fruit disease that adversely impacts oil quality, is caused by Colletotrichum species. A dominant Colletotrichum species and several secondary species have been identified in each olive-growing region. This study surveys the interspecific competition between C. godetiae, dominant in Spain, and C. nymphaeae, prevalent in Portugal, to shed light on the cause of this disparity. When Petri-dishes of Potato Dextrose Agar (PDA) and diluted PDA were co-inoculated with spore mixes produced by both species, C. godetiae displaced C. nymphaeae, even if the percentage of spores in the initial spore mix inoculation was just 5 and 95%, respectively. The C. godetiae and C. nymphaeae species showed similar fruit virulence in separate inoculations in both cultivars, the Portuguese cv. Galega Vulgar and the Spanish cv. Hojiblanca, and no cultivar specialization was observed. However, when olive fruits were co-inoculated, the C. godetiae species showed a higher competitive ability and partially displaced the C. nymphaeae species. Furthermore, both Colletotrichum species showed a similar leaf survival rate. Lastly, C. godetiae was more resistant to metallic copper than C. nymphaeae. The work developed here allows a deeper understanding of the competition between C. godetiae and C. nymphaeae, which could lead to developing strategies for more efficient disease risk assessment.

Azepane quaternary amino acids as effective inducers of 3(10) helix conformations.

A simple method for the synthesis of an azepane quaternary amino acid in enantiopure form is described. Theoretical, NMR, and X-ray studies indicated that this azepane-derived amino acid is an effective stabilizer of 3(10) helical structures in short peptides.

Cyclic amino acid linkers stabilizing key loops of brain derived neurotrophic factor.

Based on ß-turn-like BDNF loops 2 and 4, involved in receptor interaction, cyclic peptide replicas were designed, synthesized and tested. In addition to the native turn residues, the cyclic peptides include a linker unit between the N- and C-termini, selected by molecular modeling among various non-proteinogenic cyclic amino acids. NMR conformational studies showed that most of the cyclic peptides were able to adopt turn-like structures. Several of the analogues displayed significant inhibition of the BDNF-induced TrkB receptor phosphorylation, and hence could be useful templates for developing improved antagonists for this receptor.

Quaternary α,α-2-oxoazepane α-amino acids: synthesis from ornithine-derived ß-lactams and incorporation into model dipeptides.

To explore further the chemistry of amino acid-derived ß-lactams, their conversion to α,α-heterocyclic quaternary amino acid derivatives is investigated. The latter derivatives, containing 2-oxoazepane as the α,α-substituent, are synthesized by a simple Pd-C-catalyzed hydrogenolysis of Orn(Z)-derived 2-azetidinones. The rearrangement from four- to seven-membered lactam ring is driven by the key intramolecular opening of the 1-Boc-ß-lactam, initiated by 7-exotrig ring closure from the NH(2) of the Orn side chain. The synthetic route is applied to the stereoselective preparation of enantiomerically pure 4-amino-3-methyl-2-oxoazepane-4-carboxylate derivatives, for which the structure and configuration is confirmed by X-ray diffraction. Molecular modeling and NMR experiments indicate that these quaternary amino acids are able to drive the adoption of ß-turn secondary structures when incorporated in model dipeptide derivatives.

TRPV1 modulators: structure-activity relationships using a rational combinatorial approach.

A discrete library of linear and hydantoin-containing dipeptide derivatives, based on the Lys-Trp(Nps) scaffold, was prepared by solid-phase synthesis. SAR studies indicated that potency for TRPV1 blockade and selectivity towards NMDA is mainly dictated by the side-chain length and the basic nature of α, ω-groups in the N-terminal residue. The 2-Nps moiety at position 2 of Trp indole ring is preferred over the 2-pyridine one.

Synthesis and SAR studies on azetidine-containing dipeptides as HCMV inhibitors.

SAR studies on an azetidine-containing dipeptide prototype inhibitor of HCMV are described. Three series of structurally modified analogues, involving substitutions at the N- and C-terminus, and at the C-terminal side-chain were synthesized and evaluated for antiviral activity. Aliphatic or no substituents at the C-carboxamide group, an aliphatic C-terminal side-chain, as well as a benzyloxycarbonyl moiety at the N-terminus were absolute requirements for anti-HCMV activity. The conformational restriction induced by the 2-azetidine residue into the dipeptide derivatives, identified by (1)H NMR as a γ-type reverse turn, seems to have influence on the activity of these molecules.

2-alkyl-2-carboxyazetidines as γ-turn inducers: incorporation into neurotrophin fragments.

Conveniently substituted 2-alkyl-2-carboxyazetidine amino acids have been incorporated into NGF and NT3 tetrapeptide sequences to investigate their utility as reverse turn inducers (γ- vs. ß-turns). Despite the presence of an Asp residue at i position, highly preferred in ß-turns, molecular modeling and NMR studies indicated that the azetidine-containing peptides mainly stabilized γ-turn conformations.

Further evidence for 2-alkyl-2-carboxyazetidines as gamma-turn inducers.

Reverse turns, a common motif in proteins and peptides, have attracted attention due to their relevance in a wide variety of biological processes. In an attempt to artificially imitate and stabilize these turns in short peptides, we have developed versatile synthetic methodologies for the preparation of 2-alkyl-2-carboxyazetidines and incorporated them into the i + 1 position of model tetrapeptides, where they have shown a tendency to induce gamma-turns. However, to ascertain the general utility of these restricted amino acids as gamma-type reverse turn inducers, it was then required to study the conformational preferences when located at other positions. To this end, model tetrapeptides R-CO-Ala-Xaa-NHMe, containing differently substituted azetidine moieties (Xaa = Aze, 2-MeAze, 2-BnAze) at the i + 2 position, were synthesized and subjected to a thorough conformational analysis. The theoretical and experimental results obtained, including the X-ray diffraction structure of a dipeptide derivative containing this skeleton, provide evidence that the 2-alkyl-2-carboxyazetidine scaffold is able to efficiently induce gamma-turns when incorporated into these short peptides, irrespective of their localization in the peptide chain.

Synthesis and biological properties of beta-turned Abeta(31-35) constrained analogues.

A series of constrained pentapeptide analogues of the fragment Abeta(31-35) has been prepared using solid phase synthesis protocols. The results of conformational studies and surface plasmon resonance (SPR) experiments seem to indicate that the affinity of these constrained analogues for immobilized Abeta(25-35) peptide could be related to their ability to adopt a Leu34N-Ile31O beta-turn-like folded conformation.

Exceptional stereoselectivity in the synthesis of 1,3,4-trisubstituted 4-carboxy beta-lactam derivatives from amino acids.

[reaction: see text] The base-promoted cyclization of optically pure N-(p-methoxybenzyl)-N-(2-chloro)propionyl amino acid derivatives resulted in a diastereo- and enantioselective approach to valuable 1,3,4,4-tetrasubstituted beta-lactams. The stereochemical outcome of the reaction is exclusively governed by the configuration of the N-(2-chloro)propionyl moiety.

Conformationally constrained CCK4 analogues incorporating IBTM and BTD beta-turn mimetics.

To test whether a turnlike arrangement is involved in the bioactive conformation of CCK4 analogues upon CCK1 receptor recognition, we describe the preparation of two series of CCK4 derivatives, in which the central dipeptide Met-Asp has been replaced by recognized beta-turn mimetics {(2S,5S,11bR)- and (2R,5R,11bS)-2-amino-5-carboxy-3-oxo-2,3,5,6,11,11b-hexahydro-1H-indolizino[8,7-b]indole (IBTM) and beta-turn dipeptide, 2-oxo-7-thio-1-azabicyclo[4.3.0]nonane (BTD)}. The incorporation of the indolizinoindole IBTM type II beta-turn mimetic is preferred over its type II' counterpart for efficient CCK1 receptor recognition, while BTD derivatives were completely inactive. The structure-conformation-activity relationship study in the IBTM series has shown some essential requirement of these CCK4 derivatives to favorably interact with CCK1 receptors: (a) the adoption of turnlike conformations, (b) the presence of an L-Phe residue and a C-terminal carboxamide moiety, and (c) the indole ring of the IBTM skeleton. Moreover, the existence of pi-pi interactions between the phenyl ring of d-Phe residues and the indole ring of IBTM framework is detrimental for binding affinity. A series of potent and selective CCK1 receptor antagonists, exemplified by compounds 8a and 8b, emerges among these IBTM-containing derivatives.

From 1-acyl-beta-lactam human cytomegalovirus protease inhibitors to 1-benzyloxycarbonylazetidines with improved antiviral activity. A straightforward approach to convert covalent to noncovalent inhibitors.

Starting from the structure of known beta-lactam covalent human cytomegalovirus (HCMV) protease inhibitors and from the knowledge of the residues implicated in the active site of this enzyme, we designed a series of phenylalanine-derived 2-azetidinones bearing a 4-carboxylate moiety that could be apt for additional interactions with the guanidine group of the Arg165/Arg166 residues of the viral protease. Some compounds within this series showed anti-HCMV activity at 10-50 muM, but rather high toxicity. The presence of aromatic 1-acyl groups and a certain hydrophobic character in the region of the 4-carboxylate were stringent requirements for anti-HCMV activity. To go a step ahead into the search for effective HCMV medicines, we then envisaged a series of noncovalent inhibitors by simple deletion of the carbonyl group in the beta-lactam derivatives to provide the corresponding azetidines. This led to low micromolar inhibitors of HCMV replication, with 17 and 27 being particularly promising lead compounds for further investigation, although their toxicity still needs to be lowered.

Combination of molecular modeling, site-directed mutagenesis, and SAR studies to delineate the binding site of pyridopyrimidine antagonists on the human CCK1 receptor.

A rational combination of site-directed mutagenesis studies, structure-activity relationships, and dynamic-based docking of pyridopyrimidine-derived CCK1R antagonists into a refined three-dimensional model of the CCK1R allowed us to identify the receptor residues and the ligand functional groups implicated in the molecular recognition process. Our results provided unambiguous evidence that the binding site of these antagonists is overlapping that of the C-terminal tetrapeptide of CCK. In particular, Asn333 and Arg336 residues of the CCK1R are essential for high-affinity binding of these ligands. Moreover, the 2-aryl group in the pyridopyrimidine derivatives shares the same binding pocket as the C-terminal Phe side chain of CCK. Our [pyridopyrimidine.CCK1R] complex model is consistent with previous suggestions concerning the molecular basis that governs functional activity and provides useful considerations about the high CCK1 versus CCK2 selectivity of our derivatives and could contribute to fine-tune the rational design of new molecules with optimized properties.