In vitro antagonistic relationships of co-occurring phytopathogenic and non-phytopathogenic fungi associated with guava fruits.
Fungi are the most important plant pathogens since every plant is susceptible to at least one fungal pathogen and most plants are susceptible to many. Nowadays, studies of microorganism from tropical plant species are recently becoming more frequent, since they have been currently considered as potential sources of bioactive secondary metabolites for medical, agricultural and industrial development . All fungi most often interact directly or indirectly through neutralism, commensalism, mutualism, competition, parasitism and synergism, in order to survive in their habitat . As cited by Bhatia et al. , the presence of the two pathogens on the same host plant organ limits the disease development and their reproduction. On the other hand, some species of fungal endophytes play important role in host protection against predators and pathogens functioning as growth enhancers, and enable the host survival under unfavourable conditions. Also, fungi exhibit neural association with the host plants which could neither harm nor benefit the host [4, 5].
Antagonistic interaction is a natural phenomenon causing inhibitory effect to other organism which can be utilized as biological control of plant pathogen. Microorganisms as biological control agents have high potential to control plant pathogens as replacements of agrochemicals and implicate no significant effect on the environment and other non-target organisms [6, 7]. Also, as revealed by Dubey & Maheshwari , Khara & Hadwan  antagonistic association between organisms plays as a biological balancing medium of nature which includes any activity of one organism that in some way adversely affects another growing in association with it through antibiosis, competition and exploitation. Moreover, potential organisms with antagonistic capabilities include non-pathogenic bacteria, yeast, and fungi which could be cultivated in a wide array of agricultural substrates in the likes of rice, cassava, wheat bran and other valuable crops . Consequently, several researches revealed that antagonistic effect of fungi could be due to mechanisms of volatile metabolites and hyperparasitism .
As stated by Langner et al. [ 12] Fusarium, Aspergillus and Lasiodiplodia are among the most common emerging fungal pathogens in tropical and sub tropical areas with high survival and great toxicity. They are also economically important since many of its species are responsible for plant diseases including anthracnose, wilt and fruit and root rot. In addition, they are producers of secondary metabolites and mycotoxins (aflatoxins, ochratoxin, fusaric acid and fumonisin, aspergillic acid, hydroxyaspergillic acid, oxalic acid, terreic acid which are highly toxic and potential carcinogens [13, 14]. The genus Fusarium contains a number of soilborne species with worldwide distribution and known to be important and most destructive plant pathogens . And in a study of Kaur et al.  strains of non phytopathogenic Fusarium were combined with other biocontrol agents to obtain an effective biocontrol of plant pathogens. They are also commercially available in talc and charcoal based media and commercial formulations.
In recent years, antagonists have been used as biocontrol of postharvest diseases of economically important agricultural products. Thus, this study was carried out to confirm the antagonistic ability of the non phytopathogenic fungi associated with crown rot of guava which could further lead to their utilization as biological control agents against phytopathogenic fungi.
MATERIALS AND METHODS
Dual culture of non-phytopathogenic and phytopathogenic fungi was done to determine antagonistic relationship of fungi co occurring in crown rot of guava fruits.
Preparation of agar plug:
By agar plug technique, 15 ml of Potato Dextrose Agar was poured into sterile plates and allowed to solidify. Fungal isolates were inoculated individually in the prepared culture media and was incubated at 28-30 [degrees]C for seven days. Then mycelia of fungal isolates were punched with a 10 mm cork borer to form a disc of agar plug.
Dual culture assay:
Fungal discs were inoculated aseptically in PDA plates containing. The phytopathogenic was inoculated on one side of the plate and the non-phytopathogenic fungal isolate on the opposite side. For the control group, the same species of fungal isolates was inoculated juxtaposed each other. Pure cultures of fungal isolates were inoculated equidistant from the center and to the edges of the plate. Cultures were then incubated at 28-30 [degrees]C for seven days. Plates were examined from time to time until development was attained.
Identification of the Interaction:
Identification of the antagonistic relationship between the phytopathogenic and non-phytopathogenic species was based on the mycelial growth of the fungi through cultural observation and characterization. Antagonistic relationship was classified as no impact, fungistatic antagonism, territorial antagonism and mutual antagonism .
RESULTS AND DISCUSSION
Critical in the structure of fungal communities are the outcomes of interactions that develop between mycelia occupying or attempting to establish themselves on the same resources. Studies on culture indicates number of possible outcomes to interaction between species, ranging from stimulating to one or both of the mycelia, through mutual tolerance and degrees of intolerance, to the development of highly aggressive forms of competitive behavior . According to Cooke & Reiyner  when mycelia of different species are paired in laboratory, responses may be apparent either prior to contact between them or may occur only after contact has been made and may be sensitive to species-specific antagonism.
As indicated in Table 1, dual culture of Lasiodiplodia theobromae and Fusarium sambucinum with five species of Aspergillus resulted to fungistatic antagonism, wherein the non-phytopathogenic fungi were the aggressors and the phytopathogenic molds were the victims. Also, it is evident that the mycelium of the aggressor advances in a broad front over the mycelium of the victim. These findings can be attributed to the rapid growth and production of metabolites of L. theobromae (Figs 1A-E) and F. sambucinum (Figs 1F-J). It is also in agreement to Dix & Webster , wherein organisms which are more successful in competing for available substrates will prevent supplies reaching others and thus will create an antagonism. Antagonistic fungi will then flourish and eliminate those that compete due to nutritional stress. On the other hand, the inhibition of growth of phytopathogenic fungi by the non-phytopathogenic fungi can also be attributed to the possible release of volatile metabolites that diffused through the media . With this, antagonists can act as biocontrol agent through competition, antibiosis, and parasitism well as activating host defense mechanisms  .
Profuse and prolific mycelial growth of L. theobromae overgrew and suppressed the growth of the phytopathogenic fungi (A. fumigatus, A. niger, A. flavus, A. tamarii and A. japonicus). In addition, thinning on the edge and margin of the mycelia as well as decreased in sporulation of all the phytopathogenic fungi was exhibited (Figs 1A-E). Similarly, Monte  cited that most species of antagonistic fungi are fast growing, prolific spores producers and secrete antibiotics thus making them ecologically successful occurring ubiquitously in nature. In addition, phytoalexins produced by some endophytic fungi of various plants are reported to have inhibitory activity against secondary colonists and could confound antagonistic relationship among pathogens . Moreover, Rubini et al.  reported that species of genus Fusarium and Lasiodiplodia are among the endophytic fungi with antagonistic potentials inhibiting Crinipellis perniciosa, causal agent of Witches' Broom Disease. Finally, these endophytic fungi have already been recognized as source of secondary metabolites with beneficial biological activities .
Similarly, profuse and rapid growth of F. sambucinum was observed. F. sambucinum overgrew the phytopathogenic fungi resulting to its fungistatic antagonistic association with the latter. Zone of inhibition was formed between the mycelial margin of the victim (A.niger, A. tamarii, A.flavus and A. fumigatus) and the aggressor (F. sambucinum). Hence, preventing mycelial contact and overlap of the opposing fungi (Figs 1F-J). Likewise, Duffy  and Lutz et al.  also revealed the antagonistic ability of species of Fusarium in suppressing the growth of pathogenic fungi such as Trichoderma harzianum.
As reported by Chakraborty et al. , Prince et al.  and Gomathi & Ambikapathy , the formation of zone of inhibition between the two opposing fungi could be a result of mycelial response to pH alteration, competition for nutrients, enzyme substrates, oxygen, space, production of secondary metabolites and antibiotic substances either by the pathogen against antagonistic fungi or otherwise. Also, the formation of zone of inhibition is an indication for the production of antibiotic substances either by the pathogen against antagonistic fungi or vice versa . Meanwhile, hyphae of different species may intermingle in culture with no perceived benefits or disadvantage to either mycelium. Then fungistatic effect will occur constructing defensive hyphal barriers at the mycelial fronts to limit the advance of mycelium wherein the opposing mycelia will eventually stop growing even after contact between the two mycelia has been established . Most of fungal antagonistic have been used because of their high antifungal properties .
Mutualistic antagonism and fungistatic antagonism were observed between F. verticillioides and the phytopathogenic fungi (Figs 1K-O). Fungistatic antagonism resulted when F. verticillioides was paired with A. niger and A. tamarii (Figs 1 K & L). However, in this association F. verticillioides was the victim while A. niger and A. tamarii were the aggressors. Whereas mutual antagonism was observed in A. flavus, A. japonicus and A. fumigatus (Figs 1 M-O). Zone of inhibition was also observed near the center of the plates, suppressing the growth of the interacting fungi. In addition, mycelial thinning and lesser sporulation were constantly observed at the edge and margin near the opposing fungi. Furthermore, antagonistic ability of the A. niger and A. tamarii also suggest their phytopathogenic ability causing crown rot in guava fruits.
These findings are in accordance with Cooke & Reiyner  that the growth inhibition at a distance is due to the release of fungi toxic or fungistatic metabolites. Similarly Dix & Webster  mentioned that this chemical signals between combatants in mutually inhibiting pairing can lead to accumulation of mutually inhibiting product which may slow or stop the growth of competitors from some distance away. Morever, fungistatic antagonistic association of A. niger and A. tamarii with F. verticillioides coincides with the report of Gachomo & Kotchoni  and Howell  that several species of Aspergilli were inhibitory and effected the growth of several plant pathogens. Likewise, in a study of Dwivedi & Enespa , the antagonistic effect of A. niger, A. flavus, A. sulphureus, A. luchuensis completely inhibited growth the Fusarium species. Also, studies by Israel & Lodha , Simoes & Tornisielo , Gomathi & Ambikapathy  revealed the antifungal activity of A. japonicus A. flavus, A. fumigatus, A.niger, and A. versicolor against various phytopathogens. Finally Misra  and Misra & Prasad  found out that A. niger was the fastest growing and most effective antagonist against species of Fusarium causing wilt in guava.
According to Spadaro et al.  and Sharma et al.  as cited by De Medeiros , the modes of action by which antagonists suppress postharvest disease is still uncertain due to complex interactions between host, pathogen, antagonist and other microorganisms present. Possible biocontrol mechanism include the production of antibiotics, chitinolytic enzymes, direct parasitism, mycoparasitism, production of inhibitory substances and induction of resistance in the host tissue, competition for nutrients and space.
[FIGURE 1 OMITTED]
The results of the present study confirmed the antagonistic ability of L. theobromae, F. Sambucinum and F. verticilioides against A. niger, A. fumigatus, A. flavus, A.japonicus and A. tamarii. These suggest the potential of the non phytopathogenic fungi as biological control agents against the phytopathogenic fungi which would lead further to the isolation and utilization of the bioactive/ fungistatic compounds present in the tested fungal isolates.
To God be the glory. Thy will be done.
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Mary Jhane G. Valentino, Federico G. Pineda and Monina M. Fandialan
Faculty, Department of Biological Sciences, College of Arts and Sciences, Science City of Munoz, Nueva Ecija, Philippines, 3120
Address For Correspondence:
Mary Jhane G. Valentino, Faculty, Department of Biological Sciences, College of Arts and Sciences, Central Luzon State University, Science city of Munoz, Nueva Ecija, 3120
Received 12 January 2016; Accepted 28 February 2016; Available online 10 April 2016
Table 1: Antagonistic interrelationship of fungal isolates. Non- Phytopathogenic Fungi Interaction Phytopathogenic Fungi Lasiodiplodia Aspergillus Fungistatic antagonism theobromae (+) fumigatus (-) Lasiodiplodia Aspergillus niger Fungistatic antagonism theobromae (+) (-) Lasiodiplodia Aspergillus flavus Fungistatic antagonism theobromae (+) (-) Lasiodiplodia Aspegillus tamarii Fungistatic antagonism theobromae (+) (-) Lasiodiplodia Aspergillus Fungistatic antagonism theobromae (+) japonicus (-) Fusarium Aspergillus Fungistatic antagonism sambucinum (+) fumigatus (-) Fusarium Aspergillus niger Fungistatic antagonism sambucinum(+) (-) Fusarium Aspergillus flavus Fungistatic antagonism sambucinum(+) (-) Fusarium Aspegillus tamarii Fungistatic antagonism sambucinum(+) (-) Fusarium Aspergillus Fungistatic antagonism sambucinum(+) japonicus (-) Fusarium Aspergillus Mutual antagonism verticillioides fumigatus (+) (+) Fusarium Aspergillus niger Fungistatic antagonism verticillioides(-) (+) Fusarium Aspergillus flavus Mutual antagonism verticillioides (+) (+) Fusarium Aspegillus tamarii Fungistatic antagonism verticillioides(-) (+) Fusarium Aspergillus Mutual antagonism verticillioides(+) japonicus (+) * (+) = aggressor (-) = victim
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|Author:||Valentino, Mary Jhane G.; Pineda, Federico G.; Fandialan, Monina M.|
|Publication:||Advances in Environmental Biology|
|Date:||Feb 1, 2016|
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