Introduction
The jackfruit (Artocarpus heterophyllus L.) is a tropical exotic
fruit from India which has acquired importance in the state of Nayarit for being the
main producer of Mexico (SIAP, 2019).
However, crop production is reduced by postharvest diseases. Jackfruit is mainly
affected by postharvest soft rot, caused by Rhizopus stolonifer
stage (Bautista-Baños, Bosquez-Molina &
Barrera-Necha, 2014). The most used methods for its control are chemical
fungicides, among which are Dicloran and Fludioxonil. (Singh & Sharma, 2018), which have caused alterations to the
environment and residual problems, as well as the generation of resistant strains.
Therefore, low toxicity alternatives of biological origin are sought, such as
chitosan. This biopolymer that comes from the deacetylation of chitin has become a
promising alternative due to its antifungal activity and inducer of defense
mechanisms (Gutiérrez- Martínez et
al., 2018). Likewise, the efficacy of generally recognized
as safe (GRAS) in their application in food has also been evaluated (Palou, Ali, Fallik & Romanazzi, 2016).
Among these compounds, immersion of citrus fruits in potassium sorbate solutions has
been proven to be effective against P. digitatum and P.
italicum (Montesinos-Herrero,
Moscoso-Ramírez & Palou, 2016; Smilanick, Mansour, Gabler & Sorenson, 2008). These compounds are
attributed to the activation of defense mechanisms in the fruit as a consequence of
a specific recognition process between the fruit and the pathogen. Among the main
oxidation-reduction enzymes involved in these signaling processes are peroxidase
(POD) and polyphenoxidase (PPO) (Berúmen-Varela,
Coronado-Partida, Ochoa-Jiménez, Chacón-Lopez & Gutiérrez-Martínez,
2015). Peroxidases are enzymes that contribute to induced resistance by
generating reactive oxygen species (such as O2 and H2O2) that have antifungal
activity against the attack of different phytopathogens (Peng & Kuc, 1992). POD has the function of oxidizing
phenolic compounds and lignify the cell wall in plants (Yin et al., 2013). On the other hand, the PPO
enzyme is in charge of catalyzing the oxidation of phenolic compounds to quinones,
which are antimicrobial compounds toxic to the pathogen (Soliva, Elez, Sebastián & Martín, 2000). In addition, this
enzyme is involved in the lignification of plant cells favoring defense against
pathogens (Chen et al.,
2014). Therefore, the objectives of this research were to i) evaluate the
effects of chitosan (Chi), potassium sorbate (PS), and their combination on the
in vitro growth and development of R.
stolonifer, ii) to test the effectiveness of those treatments on soft
rot severity, and iii) the POD and PPO activity in jackfruit treated fruits.
Materials and methods
Phytopathogen
R. stolonifer was isolated and identified from diseased stored
jackfruits (Artocarpus heterophyllus L.) collected in San Blas,
Nayarit, Mexico.
Treatments
Chitosan (75-85% deacetylation) (Sigma-Aldrich, St. Louis, MO, USA) was prepared
in concentrations of 0.1, 0.5 and 1.0% (w/v) based on the methodology described
by Ramos- Guerrero et al. (2018). Potassium sorbate solutions
(Jalmek, Mexico) were prepared in concentrations of 1.0, 1.5, 2.0, 2.5 and 3.0%
(w/v) in sterile distilled water. Once the antifungal effectiveness of the
separate treatments was determined, treatment combinations of 1.0% Chi + 0.1%
PS, 1.0% Chi + 0.5% PS and 1.0% Chi + 1.0% PS were selected, according the most
effective concentrations in preliminary trials.
In vitro evaluations on R. stolonifera
The mycelial growth of the pathogen was evaluated by taking a 10 mm diameter disk
from the edge of the colony. The disk was placed in the center of the Petri dish
with PDA amended with each of the concentrations and/or combinations previously
described. Petri dishes with only PDA were used as controls. Petri dishes were
incubated at 25 ºC and the mycelial growth diameter was measured with a digital
vernier (TruperTM) every 24 h until the pathogen completely colonize the Petri
dishes in the control. The results were expressed in radial growth in mm of the
circumference of the fungus in the Petri dish, as well as in percentage of
inhibition of mycelial growth, comparing the treatments with the controls. The
sporulation test was carried out after 48 h of incubation of the fungus using
the same Petri dishes with the treatments and the control of the mycelial growth
assay, using the methodology described by Cortes-Rivera, Blancas-Benitez,
Romero-Islas, Gutiérrez-Martinez & González-Estrada (2019). In the spore
germination assays, aliquots of 20 μL were taken from a spore suspension of 1 ×
106 conidia mL− 1, which was placed on discs with the different treatments of
Chi, PS, Chi-PS, and only PDA as a control. Finally, around 400 spores were
observed in a microscope Motic BA 300 (Motic Instruments Inc., Canada) every
hour for 6 h at 40X. The results were expressed as the percentage of germination
compared to the control.
In vivo evaluation in jackfruit
The jackfruit was harvested at physiological maturity in San Blas, Nayarit,
Mexico, and then transported to the food biotechnology laboratory of the TecNM /
Technological Institute of Tepic. Fruits were washed with a 2% (v / v) sodium
hypochlorite solution (NaClO) and left to dry at room temperature. Next, 30 µLof
a 1 x 106 spore/mLspore suspension were inoculated by wound using a sterile
needle (2.5 mm deep and 3 mm wide). Fruits were allowed to dry for 30 min and
then the jackfruits were subjected to spray treatment with a Chi-PS solution,
allowed to dry at room temperature for 60 min and then were stored at 24 ºC in
humid chambers (80% humidity) for 96 h. Finally, the disease severity was
evaluated according to Velázquez-del Valle,
Bautista-Baños, Hernández- Lauzardo, Guerra-Sánchez & Amora-Lazcano
(2008). Control fruits were sprayed with water.
Enzyme activity
The enzyme activity was evaluated at 0, 24, 48, and 72 h after the application of
the treatment Chi (1.0%) + PS (1.0%) in jackfruit fruits at physiological
maturity. The enzymatic extract was obtained from the cuticle of the jackfruit
using the methodology described by Chen Bélanger,
Benhamou & Paulitz (2000). POD expression was evaluated using the
technique proposed by Chance & Maehly
(1955) with some modifications. Briefly, 0.5 mL of crude extract
(supernatant) were mixed with 2 mL of a guaiac buffer solution, and then
incubated for 5 min at 30 ºC. Subsequently, 1 mL of H2O2 was added to the
mixture and the absorbance was measured at 460 nm every 5 s for 90 s in a UV /
visible spectrophotometer (JENWAY 67 series). On the other hand, PPO activity
was determined according to Yue-Ming
(1999). Briefly, 0.5 mL of crude extract were mixed with 3 mL of
catechol (as substrate) and then the absorbance was measured at 420 nm every 10
s for 180 s. The activity of both enzymes was expressed as U mg protein-1. The
protein content was determined by the Bradford
method (1976). Data were analyzed by analysis of variance (ANOVA)
with a 5% level of significance using a completely randomized block design. A
comparison of means was performed by Tukey’s test when the ANOVAshowed
significant differences. The statistical package IBM SPSS statistics 25 was
used.
Results and discussion
The Mycelial growth inhibition, sporulation, and germination are shown in Table I, observing a significant difference in
all the treatments compared to the control. In 48 h, the control has a growth of
80.0 mm. The lowest mycelial growth was observed at a concentration of 1.0% of Chi
(41.6 mm), obtaining a 48% inhibition of mycelial growth. The inhibition of the
sporulation of R. stolonifer was observed from the 0.1% treatment,
obtaining a lower concentration of spores with the 1.0% treatment (1.28 x 105 spores
mL-1). Regarding the germination of spores, the germination in the
PDA discs was inhibited with the treatments up to 60% in the 1.0% concentration of
the biopolymer compared to the one that presented 100% at 4 h. The parameters
evaluated of the PS at different concentrations are shown in Table I. One % PS inhibited mycelial growth by 70%, while
complete inhibition was observed at 1.5. In the sporulation test, the solution with
a concentration of 3.0% of PS presented the lowest number of spores (2.1 x 105
spores mL-1). Table I shows the
results of the PS concentrations used in this experiment, observing a statistically
significant control (0.0%) in the germination of spores with a concentration of
1.0%, which was monitored for up to 6 h. In Table
I, it can be seen that the combination of Chi and PS treatments
completely inhibited mycelial growth from the lowest concentration of (0.1% Chi-1.0%
PS). The mycelial growth inhibition and germination of R.
stolonifer isolated from the jackfruit are due to the synergistic
effect of chitosan and sodium sorbate. Taking this into account, chitosan is
attributed to its polycationic nature, since its molecule is positively charged by
the presence of amino groups, which interact with the negative charges of the cell
wall of the microorganism, achieving a break in its structure, carrying out the loss
of protein compounds and intracellular constituents (Ayala Valencia, 2015). Regarding the effect of potassium sorbate, Smilanick et al. (2008)
describe that this compound generates alterations in the structure of the cell as
well as alterations in the cell membrane and the inhibition of enzymes that are
involved in metabolism in the transport functions. In previous studies, the
affectivity of chitosan added with potassium sorbate to inhibit the mycelial growth
of P. citrinum isolated from garlic has been reported with an
effectiveness of 99.5%, attributing it to the decrease in intracellular pH and
ionization by of K+ in the chemical structure of PS, affecting the development of
the fungus (González-Estrada et
al., 2020).
Table I
Effect of Chi, PS, and Chi-PS at different concentrations on mycelial
growth, percentage of mycelial growth inhibition, sporulation, and
percentage of germination of R. stolonifer.
Treatment |
Mycelial growth (mm) (48 h) |
Mycelial growth inhibition (%) |
Sporulation (spores/ mL) |
Germination (6 h) (%) |
Control
|
80.0 ± 0.20 a |
0 ± 0.0 a |
143.2x106 ± 0.15 a |
100 ± 0.0 a |
Chi 0.1 % |
60.0 ± 0.38 b |
25 ± 0.28 b |
4.24x105 ± 0.09 b |
80 ± 0.3 b |
Chi 0.5 % |
44.8 ± 0.24 c |
44 ± 0.35 c |
3.06x105 ± 0.03 c |
75 ± 0.4 c |
Chi 1.0 % |
41.6 ± 0.32 d |
48 ± 0.23 d |
1.28x105 ± 0.04 d |
60 ± 0.5 d |
Control
|
80 ± 0.2 a |
0 ± 0.0 a |
143.2x106 ± 0.15 a |
100 ± 0.0 a |
PS 1.0 % |
24 ± 0.22 b |
70 ± 0.3 b |
12x106 ± 0.82 b |
0.0 ± 0.0 b |
PS 1.5 % |
0.0 ± 0.0 c |
100 ± 0.0 c |
8x105 ± 0.35 c |
0.0 ± 0.0 b |
PS 2.0 % |
0.0 ± 0.0 c |
100 ± 0.0 c |
8x105 ± 0.52 c |
0.0 ± 0.0 b |
PS 2.5 % |
0.0 ± 0.0 c |
100 ± 0.0 c |
5.6x105 ± 0.48 d |
0.0 ± 0.0 b |
PS 3.0 % |
0.0 ± 0.0 c |
100 ± 0.0 c |
2.1x105 ±0.32 e |
0.0 ± 0.0 b |
Control
|
80.0 ± 0.2 a |
0 ± 0.0 a |
143.2x106 ± 0.15 a |
100 ± 0.0 a |
0.1% Chi-1.0% PS |
0.0 ± 0.0 b |
100 ± 0.0 b |
0.0 ± 0.0 b |
0.0 ± 0.0 b |
0.5% Chi-1.0% PS |
0.0 ± 0.0 b |
100 ± 0.0 b |
0.0 ± 0.0 b |
0.0 ± 0.0 b |
1.0% Chi-1.0% PS |
0.0 ± 0.0 b |
100 ± 0.0 b |
0.0 ± 0.0 b |
0.0 ± 0.0 b |
In vivo evaluation
Once the best treatments were established, combination treatment with Chi (1.0%)
- PS (1.0%) was applied in the jackfruit fruits. The jackfruit inoculated with
R. stolonifer and treated only with water showed an
accelerated infection, detecting signs of rot at 48 h after inoculation. In
addition, at 96 h the control presented 100% infection (Figure 1D). An accelerated softening of the fruit was also
observed, evidence of the advance of the ripening process. Further, in the
control treatment (fruits with natural infection) (Figure 1B), the development of soft rot symptoms was visualized in
different areas of the fruit. At 96 h, the fruit was completely deteriorated by
the fungus. The fruits treated with Chi-PS (with and without inoculation of the
pathogen) had a 0.0% severity of the infection (Table II). The effectiveness of the Chi-PS treatment on the
jackfruit can be due to the influence of the pH of the compound since it has
been reported that its application on the cuticle of certain citrus fruits,
reporting a value of 5.5 and when a wound occurs, it can drop to 5.1. The
application of the treatment regulates this value, maximizing the activity to
inhibit the invasion of the pathogen (Smilanick
et al., 2008). On the other hand, it has been
described that chitosan generates a modified atmosphere in the fruit, regulating
its maturation and senescence processes, preventing the development of pathogens
that could infect the fruit after harvest, as well as the potential to induce
enzymes against the attack of pathogens (Bautista-Baños, Ventura-Aguilar, Correa- Pacheco & Corona-Rangel,
2017), and phenolic compounds in plants (Benhamou, 1996).
Figure 1
Jackfruit fruits with the different treatments: (A) No inoculated
at 0 h, (B) No inoculated at 96 h, (C) Inoculated at 0 h, (D)
Inoculated at 96 h, (E) No inoculated treated with Chi-PS at 0 h,
(F) No inoculated treated with Chi-PS at 96 h, (G) Inoculated
treated with Chi-PS at 0 h and (H) Inoculated treated with Chi-PS at
96 h. Source: self-made.
Table II
Disease severity in jackfruit fruits treated with chitosan and
potassium sorbate with and without inoculation of Rhizopus
stolonifer.
Treatment |
Disease severity (%) |
Control |
100 ± 0.0 a |
Control (inoculated) |
100 ± 0.0 a |
Chi + PS |
0 ± 0.0 b |
Chi + PS (inoculated) |
0 ± 0.0 b |
Enzyme activity
The effect of Chi and PS (with and without inoculation of the pathogen)
maintained a high enzymatic activity compared to control in which it was
relatively low. In the fruits inoculated and sprayed with the combined treatment
(Chi-PS) at 24 h the highest level of activity was presented by this enzyme
subsequently decreasing in relation to the control (p <
0.001) (Figure 2 A),
as well as the non-inoculated fruits showed higher activity at 72 h after
treatment application. In Figure 2 B, the
activity of the PPO is presented, recording an increase in its activity as time
passes, observing a difference (p < 0.001)
between the controls (with inoculation and without inoculation) and the fruits
treated with the solutions of chitosan and potassium sorbate, showing greater
activity at 48 h after their application. It is observed that in the treated
fruits, the activity of the enzyme (Figure
2B) was induced from 12 h and according to time it increased,
observing that at 24 h it reached its maximum induction. The observed increases
in POD activity seem to be related to the pathogen to the fruit, stimulating a
series of mechanisms in the synthesis of reactive oxygen species such as
superoxide radicals and hydrogen peroxide, acting as a signal, and regulating
gene expression and strengthening the cell wall via protein cross- linking
(Blechert et al.,
1995). These results agree with those obtained by Liu, Tian, Meng & Xu (2007) in tomato
fruits, observing that enzymatic activity of POD was increased at chitosan 1.0%
concentration, stored at 25 ° and 2 °C. POD and PPO enzymes are part of a
defense system in plants against stress situations generated by the invasion of
pathogens (García-Garrido & Ocampo,
2002; Kazan, Murray, Goulter,
Llewellyn & Manners, 1998). Berumen-Varela et al. (2015), observed that the
1.0% chitosan induced the enzymatic activity of POD in mango fruits, observing
the highest activity at 24 h (without inoculation) and 72 h (with inoculation)
after applying the treatment. Polyphenoloxidases have been shown to catalyze the
oxidation of phenolic compounds to quinones using molecular oxygen as an
electron acceptor (Sommer, Petersen & Bautz,
1994) which are toxic to pathogens and pests (Weir, Park & Vivanco, 2004). Polyphenoloxidases have
been suggested to be directly involved in auxin biosynthesis because the
o-quinones produced can react with tryptophan to form indole-3-acetide (Jukanti, 2017). The use of GRAS compounds
can protect against infections of R. stolonifer by activating
mechanisms of fruit defense.
Figure 2
Effect of Chi-PS treatment on enzyme activity over time: (A)
polyphenol oxidase and (B) peroxidase. Values are expressed as means
± standard deviation (N = 15).
Conclusions
The combined treatment of chitosan and potassium sorbate inhibited the germination of
spores of R. stolonifer and significantly reduced soft rot in
jackfruit without damaging the quality of the fruit. Chi-PS treatmentled
toasignificant increase in the enzymatic activity of PPO and POD. These results show
that the application of these GRAS compounds is a promising method for the control
of fungal diseases in the post-harvest stage and represents an efficient, reliable
and safe method to replace fungicides.
Acknowledgments
The author’s thanks to Tecnológico Nacional de México for the financial support
(9576.20-P) and to the National Council of Science and Technology (CONACYT) for the
doctoral scholarship granted.
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