Comportamento morfofisiológico e bioquímico de cultivares de pimenteira do reino (Piper nigrum L) submetidas ao cultivo in vitro com filtrado de cultura de Fusarium solani f. sp. piperis Alb.

Abstract

The plantation of Black pepper (Piper nigrum L) is very expressive in Brazil and, particularly in the state of Pará, since it is mostly grown for exportation and its price has a remarkable variation in the international market. However, one factor which seriously affects its production is the occurrence of fusarium, caused by the fungi Fusarium solani f. sp. piperis, which occurs at epidemic levels in crop areas. This paper aimed to find possible differences among the cultivars of black pepper in vitro when submitted to filtrates obtained from the culture of F. solani f. sp. piperis, as well as to check the magnitude of the changes in growth, morphophysiology and biochemical features of P. nigrum. In order to do so, black pepper fruits, from Guajarina, Kottanadan, Balankotta and Singapore cultivars were collected and at Western Amazon Embrapa- Belém/Pará laboratory, seeds were inserted in vitro to grow the plants. At first, the mellow fruits with yellow and red colors, were collected and submitted to pre-sterilization, which comprised: seeds pulping, washing in running water and immersion in sodium hypochlorite (NaCIO) at 1.5% during the night in a greenhouse at 37ºC. The seeds were grown in vitro in medium with MS (14 MS), with 3% of saccharose, vitamins, 0.17 elL' NaH,PO; (monobasic sodium phosphate), 0.2% of phytagel. The plants which showed better formation were selected and multiplied by five sub-crops in basic medium of MS culture containing 3% of saccharose, 0.2% of vitamins and 0,2% of phytagel and supplemented with 0.5% mg.L” of 6-benzilaminopurina and 0.2 mg.L”' of indoleacetic acid. The medium pH was adjusted to 5.8 and submitted to autoclaving for 20 minutes under a pressure of 1.5 atm and at 120 ºC. The sprouts multiplied after five sub-crops were fixed onto a medium with 4 MS, 0.2% of vitamins, 3% of saccharose and 0.2% of phytagel and supplemented with 0.05 meg.L' of Naphthaleneacetic acid, and kept under such conditions for four weeks. Plants from the Guajarina cultivar were used for an initial experiment, since this cultivar is the one most grown in the state, and fungal filtrate in doses 0, 20, 30, 40 and 50% (v/v). The pH of the medium was adjusted to 5.8 and submitted to autoclaving for 20 min under pressure of 1.5 atm and 120 ºC. After 45 days from the beginning of the procedure, the morphophysiological aspects were then assessed. Morphological changes and growth decrease were observed on the Guajarina cultivar from the dose with 20% of fungal filtrate. The alterations observed were in the root length, number of roots, numbers of leaves and leaf area, proportionally reduced as long as the concentration of fungal filtrate increased. Thus, the fungal filtrate in medium in any concentration causes alteration on the in vitro growth of plants from the Guajarina cultivar. Doses with 30% or more were considered efficient to be used in other experiment. Another experiment with black pepper from four cultivars of P. nigrum (Singapore, Balankotta, Kottanadan and Guajarina), cultivated in vitro and submitted to a filtrate obtained from a culture of F. solani f. sp. piperis was done. Plants rooted onto a medium with MS (74 MS), 0.2% if vitamins, 3% of saccharose and 0,2% of phytagel and supplemented with 0.05 me.L Naphthaleneacetic acid for four weeks were transferred to test tubes containing medium of 4 MS and fungal filtrate in doses 0% and 35% (v/v), kept in growth rooms under controlled conditions with photoperiod of 16 hours with luminous intensity of 3000 lux and temperature of 25 + 2ºC, and evaluated after this period in relation to alterations in growth, biochemical and morphological features. Growth decrease and morphological changes were observed on the cultivars when submitted to the action of the fungal filtrate. The filtrate of fungal culture caused morphophysiological changes mainly in the root systems of the cultivars of black peppers in vitro. The fungal filtrate caused decreases in the total biomass production and in the relative growth rate of P. nigrum plants, except the cultivar Kottanadan. We also observed that the fungal filtrate decreases the concentration of proteins in the black pepper cultivars. Changes in the contents of glucose, fructose and starch were also noticed on the cultivars of Guajarina and Kottanadan. Therefore, the cultivars of P. nigrum present distinct morphophysiological, biochemical and growth-related responses once under action of the fungal filtrate, so that the Singapore cultivar is the most susceptible to the fungal filtrate and the least susceptible is the Kottanadan cultivar.

Black pepper is the most important spice in nowadays world trade and has a great economical importance for Brazil and for the state of Pará. However. The state productivity has a remarkable oscillation due to many factors, mainly the phytosanitary ones. The filtrate of the fungi Fusarium solani f. sp. piperis has been used to evaluate the sensibility of plants in vitro. The Fusarium disease causes several symptoms, even the plant death. This paper aimed to determine, from the changes in morphophysiology and in growth, the magnitude of such changes in plants of Guajarina cultivar in vitro under effect of a filtrate of Fusarium solani f. sp. piperis in medium. In order to do this, the seeds were grown in vitro and the resulting plants were used as sources of explants for the micropropagation process. The sprouts multiplied after five sub-crops were fixed in amedium with 42 MS, 0.2% of vitamins, 3% of saccharose and 0.2% of pPhytagel and supplemented with 0.05 mg.L of Naphthaleneacetic acid. The medium pH was adjusted to 5.8 and submitted to autoclaving for 20 minutes under pressure of 1.5 atm at 120ºC, and kept under such condition for four weeks. The plants were kept in growth room under controlled temperature, photoperiod and luminous intensity conditions. After this period, they were transferred to test tubes containing medium with 4 MS and fungal filtrate in doses O, 20, 30, 40 and 50% (vv). The plants were kept in growth-room for 45 days and then had their changes in morphology (number of leaves, number of roots and roots length) and growth (fresh and dry mass of roots and leaves) assessed. Morphological changes on plants from the cultivar Guajarina were noticed from doses with 20% of fungal filtrate. The changes noticed were in the root length, number of roots, number of leaves and leaf area, proportionaliy reduced as long as the concentration of fungal filtrate increased. Thus, the fungal filtrate in medium at any concentration causes changes on in vitro growth of plants from black pepper Guajarina cultivar. Reductions on growth are remarkable from doses with 30%. Total nitrogen in the stalk and leaves of P. nigrum was reduced once in the presence of the fungal filtrate, probably due to chlorosis and to leaf senescence. Total carbon in the plants parts with presence of fungal filtrate also increased. Doses equal or above 30% were considered efficient to be used in another experiment.

Remarkable is the economical and social expression of the black pepper cultivation in Brazil and particularly in the state of Pará. The phytosanitary problems are among the main jeopardizing factors for the state productivity. The fusarium disease stands out, since it may bring the plant to death. The filtrate of the causative agent of this disease, the fungi Fusarium solani f. sp. piperis, has been used to evaluate the susceptibility of in vitro plants. This paper aimed to assess the sensibility of four cultivars of nigrum (Simgapore, Balankotta, Kottanadan e Guajarina) grown in vitro to F. solani f. sp. piperis culture filtrates by evaluating the magnitude of morphophysiological changes related to the plants development aiming to test the hypothesis that there are different behaviors among the genetic material comprising responses to the toxins from the fungal filtrate over the physiological and growth-related parameters. To do this, seeds were grown in vitro in medium with MS (14 MS), with 3% of saccharose, vitamins, 0.17 g.L”" NaH,PO, (monobasic sodium phosphate), 0.2% of phytagel, pH adjusted to 5.8 before autoclaving at 121ºC for 20 minutes and cultivation conditions as 25 + 3 ºC and photoperiod of 16 h of light. The plants were used as sources of explants (stalk apex and nodal segments) for micropropagation. During the five sub-cultivations, the sprouts were multiplied in a basic medium with MS culture containing 3% of saccharose, vitamins and 0.2% of phytagel and supplemented with 0.05 mg.L of 6- benzilaminopurina and 0.2 mg.L” of indoleacetic acid and had the pH adjusted to 5.8 before being autoclaved for 20 minutes. The sprouts multiplied after the five sub-cultivations were fixed in a medium with 4 MS, 0.2% of vitamins, 3% of saccharose and 0.2% of phytagel and supplemented with 0.05 mg.L' of Naphthaleneacetic acid for four weeks and then transferred to test tubes containing medium with 14 MS and fungal filtrate in doses of O and 35% (v/v). The plants were kept in growth-room under controlled conditions, photoperiod of 16 hours and luminous intensity of 3000 lux and temperature of 25 + 3ºC, and then evaluated in relation to changes in morphology and growth. Morphological changes and growth decreases were noticed on the cultivars once submitted to the action of the fugal filtrate, mainly on their root systems. The fugal filtrate caused decreases on the total biomass production and on the relative growth rate of P. nigrum plants, except the cultivar Kottanadan, and also caused decrease on the proteins concentration, besides changes on the glucose, fructose and starch contents of the cultivars Guajarina e Kottanadan. Therefore, the cultivars of P. nigrum show different morphophysiological, biochemical and growth-related responses when under action of the fungal filtrate, so that the Singapore cultivar was the most susceptible to the filtrate and the Kottanadan was the least susceptible one.