Aminoacids Absorption by Poblano Chili Crops in Presence of Fulvi-75 Organic Fertilizer

Fulvi-75, made and distributed by Mexican Company Fitochem (www.fitochem.com. mx), was used as fertilizer. It is a compound made by 78.9 % of fulvic acids as percentage of final mixture. Fifteen 14C labelled aminoacids were used: 14C-L- Amino Acid Mixture, 50 μCi (1.85MBq) in ethanol: water (2.98), sterile packaged, Perkin-Elmer, Product number NEC850E. Seeds of Mexican vegetable poblano chili were used to test the absorption of mentioned aminoacids, till 21 days later they started their germination. It was considered in this research, that 14C present in the fertilizer, as natural component by its organic cycle, is negligible when compared with 14C labelled aminoacids, and it was considered as background of counts per unit time, from those counts produced by used aminoacids (Table 1), since any way they are absorbed in different proportion when fertilizer is used.

Table 1: 14C labelled aminoacids (Perkin-Elmer product number NEC850E) and percentage present in the mixture)(Total Radioactivity 50 μCi).

Trichoderma spp. are known as effective biological control agents that are isolated from soil and plants [12]. These mycoparasitic fungi are armed with different types of cell wall degrading enzymes such as chitinases [13], cellulases [14], glucanses [15], and proteases [16]. Also, they produce various volatile (such as 6-Pebtenyl Alpha Pyrone, 6-PAP) and involatile antibiotics [17], as well as a range of peptaibiotics [18] and lipases [19] that make them competent disruptors of microbial cell membranes [20] and capable of insect-cutin destruction [21]. This rich arsenal leads to their unique abilities to control a range of plant pathogenic fungi, oomycetes, gram-positive as well as gram-negative bacteria [22], nematodes [23], and even insect pest [24].The fungi produce huge amounts of green spores expected to be more UV-resistant than the hyaline spores of the entomopathogenic fungi such as Beauveria Bassiana [25]. Trichoderma spp. are easily cultivated on simple media are among potent competitors able to fast colonize various substrates [26] and niches on plants [27]. Importantly, these fungi are able to induce plant defensive system through salicylic acid, jasmonic acid and ethylene signaling pathways [28-32]. Unlike chemical inducers of plant resistance that lead to reduced crop yield as the result of matter and energy costs put for the motivation of plant defensive machinery, Trichoderma spp. also promote plant growth and yield [33-36] and increase their tolerance to heat, salinity and drought stresses [37]. Trichoderma spp. are well-known mycoremediants. Interestingly, Trichoderma spp. and other beneficial fungi such as arbuscular mycorrhizal fungi and Serendipita indica synergistically impose positive effects on plant growth and yield [26]. These fast growing filamentous fungi produce numerous number of actively secretive hyphal tips and since have extensively been applied for heterologous protein production and secretion [38]. The hyphal structure can penetrate into various solid substrates including plant residues, and the body of invaded (micro) organisms [39].

Therefore, Trichoderma spp. seem to fulfill most of the requirements in an ecofriendly agricultural system. However, some enzymatic differences have been found in alcohol dehydrogenase, acetyl CoA synthetase, pyruvate decarboxylase, and aldehyde dehydrogenase between Trichoderma reesei and the entomopathogenic fungus, Metarhizium anisopliae [40]. As the metabolic engineering seems rather difficult and time-consuming, the superior isolates of Trichoderma spp. can be genetically engineered in order to produce and secrete a range of insecticidal peptides. I have called these genetically transformed superiors as Trichodermans [25]. These are able to use their signal transduction pathway [41] in order to pursue the parasitic fungi as well as insects, grow toward more humid areas and infect the concealed evils in planta. Different insect-specific insecticidal neurotoxic peptides have been identified in arachnida (such as spiders and scorpions), cnidarians and mollusks that target specific sites on metal ion channels [42, 43]. Although, it is believed that spider insecticidal peptides globose (easy to be secreted) and more stable (resistant to proteases) [44], the co-transformation of superior T. asperelloides isolates with a genetic construct for the expression and secretion of µ-agatoxin IV (a neurotoxin from American funnel web spider, Agelenopsis aperta) using Trichoderma harzianum 42KDa endochitinase signaling peptide was not successful. The reason seemed to be the endoplasmic reticulum stress imposed due to the short length of the peptide (37 aa) and four intramolecular disulfide bonds [45]. Another reasom might be that the natural toxin is of an amidated carboxylic end and such an amidation might have not been taken place in T. asperelloides. The construct was successfully expressed (as revealed by the bicistronic expression of eGFP), however, fungal conidiation was inhibited possibly due to the ER- and the subsequent secretory-stress inhibited [25].

Figure 1 The conidiphore of Trichoderma asperelloides genetically transformed to express two separate open reading frames coding for the bicistronic co-expression of eGFP and the insecticidal peptide µ-agatoxin IV under government of a single promoter. So, the intensity of green fluorescence infers to the rate of toxin production, and allows histopathological studies on pests and pathogens due to be tested [25]. There are other insecticidal peptides that target different sites on sodium, calcium, and potassium ion channels of insect neurotic system. For example, hainantoxins and huwentoxins specifically target site-1 on votalge-gate sodium channels [46]. Alpha-insect toxins are another group of insect-specific toxins of insect origin that target site-3 on sodium pumps [47]. Interestingly, at least some of the insecticidal peptides are insect group-selective and even species-selective [48]. Making it possible to control deleterious insect pests without harmful impacts on beneficial insects. Anyway, biking toward the realization of Trichoderman can be peddled using other insecticidal peptides.

Figure 1: The conidiphore of Trichoderma asperelloides genetically transformed to express two separate open reading frames coding for the bicistronic co-expression of eGFP and the insecticidal peptide μ-agatoxin IV under government of a single promoter. So, the intensity of green fluorescence infers to the rate of toxin production, and allows histopathological studies on pests and pathogens due to be tested [25].

Two groups with same weight of poblano chili seeds were sown in equal masses of black ground land, in two different plant pots, numered 1 and 2. Once they have started to grow up, both of them were irrigated daily during 21 days with 50ml of water solution containing the mixture of aminoacids labelled with 11.1 d/ml-m of 14C, previously made by diluting 50 μCi of commercial product from Perkin Elmer, The radiochemical Centre, Amersham, England, in 1 liter of water, and then 1ml. of this solution in 100ml. of water, and finally 1ml. of this in another 100ml. of water, to get an activity of 11.1d/m-ml, used to irrigate daily pots 1 and 2, each one with 50ml. of labelled solution [1,2]. But pot 1 was irrigated also with 50ml. of one solution containing 1mg/ml of the organic fertilizer Fulvi-75, produced by Mexican industry Fitochem (www.fitochem.com.mx), while pot 2 was irrigated also with 50ml. of just water. After 21 days of daily irrigation, one little leaf from plants in pots 1 and 2 were cut out and weighed. These leaves were then crushed by separate in two small laboratory mortars, which were washed each with 4ml. of solvent used for liquid scintillation detector (Ultima Gold CR, Perkin-Elmer), and detected during 4 hours, same time considered to obtain the background counts in the system with just liquid scintillation (Figure 1).

Figure 1: Fulvi-75, 14C labelled aminoacids and small experimental plantation.

Counts obtained from leaf samples in pot 1 and 2 were corrected by background and they show one statistical variation to 1.6% and 2.3% respectively, when divided by counts per time and leaf weight. Then, result from sample 1 was divided by result from sample 2, and a final result equal to 2.76 or 276% was produced.

This result seems to proof that pot 1, irrigated with fulvic acids in water solution, it has absorbed one proportion equal to 2.76 times more labelled aminoacids than pot 2, irrigated with just water, while both pots 1 and 2 were irrigated with same dose of aminoacids.

1. Navarrete JM, Urbina VM, Martínez T, Cabrera L (2005) Autoradiography of mineral ions in green leaves and flowers, absorbed with and without synthetic fulvic acids. J Radioanal Nucl Chem 263: 779-781.
2. Anesio AM, Hollas C, Graneli W, Parry JL (2004) Influence of humic substances on bacterial and viral dynamics in fresh waters. Appl Environ Microbiol 70(8): 4848-4854.

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