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American Journal of  
Environment and Climate (AJEC)

Antifungal Effects of  Different Commercial Products Against Fusarium oxysporum 
f. sp. cubense TR4 Causing Fusarium Wilt of  Banana

Mary Amor G. Figueroa1, Artemio S. Hagonos, Jr.2

Volume 1 Issue 2, Year 2022
ISSN: 2832-403X (Online)

DOI: https://doi.org/10.54536/ajec.v1i2.207
https://journals.e-palli.com/home/index.php/ajec

Article Information ABSTRACT

Received: April 09, 2022

Accepted: May 25, 2022

Published: June 02, 2022

Banana production is seriously threatened by Fusarium wilt, also known as Panama disease 
worldwide. Regulatory control and the use of  biological control agent, Trichoderma harzianum 
showed to be effective in minimizing the development and spread of  the disease in the 
area. Some studies also showed the potential of  some commercially-available fungicides 
in managing the disease. In this study, different commercial products applied singly and in 
combinations were evaluated against FocTR4 causing Fusarium wilt of  banana and the most 
effective treatment combinations for the management of  FocTR4 was determined under 
in vitro test condition. Treatments used in the experiment were as follows: T1- Control; 
T2- B. subtilis alone; T3-Fosetyl-Aluminum alone; T4-Fluopyram alone; T5- B. subtilis + 
Fosetyl-Aluminum; T6-B. subtilis + Fluopyram; T7-Fosetyl-Aluminum + Fluopyram and T8- 
B. subtilis + Fosetyl-Aluminum + Fluopyram. The experiment was laid out in Completely 
Randomized Design (CRD) with three replications. Data were statistically analyzed using 
the Analysis of  Variance (ANOVA) and the treatment means were compared using Tukey’s 
Honest Significant Difference (HSD). Results of  the study showed that treatments of  B. 
subtilis in combinations with Fosetyl-Al or Fluopyram and the combinations of  these three 
products had totally inhibited and killed FocTR4. Single treatment of  Fosetyl-Al and the 
combinations of  the products had totally inhibited the growth and spore production of  
FocTR4 however, validation test revealed that Fosetyl-Al alone and its combination to 
Fluopyram induces chlamydospore production instead of  killing the pathogen.

Keywords

FocTR4, Bacillus subtilis, 
Fosetyl-aluminum, Floupyram

1 Plant Pathology Faculty, Surigao State College of  Technology, Mainit, Surigao del Norte, Philippines
2 Plant Pathology Faculty, University of  Southeastern Philippines, Tagum-Mabini Campus, Tagum City, Philippines.
* Corresponding author’s e-mail: mafigueroa03@gmail.com

INTRODUCTION
Global banana production is seriously threatened by 
Fusarium wilt, also known as Panama disease. The disease, 
caused by the soil-borne fungus, Fusarium oxysporum f. sp. 
cubense (Foc), wiped out the Gros Michel-based banana 
industry in Central America and the Caribbean in the 
mid-twentieth century. The effects of  Foc Race 1 were 
overcome by a shift to resistant ‘Cavendish’ cultivars, 
which are currently the source of  99% of  banana exports 
(Vicente et al., 2014).
Unfortunately, they also reported that a new race of  Foc 
called Tropical race 4 (TR4) has overcome Foc resistance 
in ‘Cavendish’ clones. Perhaps even more seriously, other 
banana cultivars such as plantains, cooking bananas and a 
diverse range of  dessert banana varieties (not susceptible 
to races 1 and 2) are also susceptible to TR4. These local 
varieties are mostly grown by smallholder farmers for 
local consumption and income generation. More than 
80% of  global banana and plantain production is thought 
to be based on TR4-susceptible germplasm. This race of  
Foc has caused epidemics in ‘Cavendish’ in the tropics 
different from those less severe infections previously 
reported in the sub-tropics.
In the Philippines, the disease was suspected to be 
present in the country since the 1970s, but TR4 was 
only confirmed in 2008. Its incidence in the surveyed 
farms increased from 700 cases in 2005 to 15,000 in 
2007 (Molina et al., 2008). Large companies currently 
manage the disease following the protocol established for 
bacterial wilt (Ralstonia solanacearum) management, which 

is based on quarantine, sanitation, soil disinfestation and 
fallows (Molina, 2009). 
Considering the prevalence, ubiquitous nature and the 
ability of  fungi to cause epidemics in relatively short 
period of  time, disease management strategies are 
needed to secure the productivity in today’s agriculture. 
Chemical control measures are particularly common 
in management of  fungal plant diseases, and most of  
these measures rely on the use of  fungicides (Ivic, 2010). 
The use of  biological control methods also offers an 
excellent alternative strategy for effective control of  
various diseases as well as augmentation of  nutrient 
availability in the rhizosphere. In fact, previous studies 
on commercially-available microbial agents applied singly 
and in combinations with synthetic products showed the 
potential of  these agents in controlling various diseases. 
In addition, for successful control of  the disease, these 
products must be delivered in an appropriate state of  
activity and to the right place at the right time in order to 
achieve economical control.
Thus, this study was conducted to evaluate the 
efficacy of  different commercial products such as B. 
subtilis, Fosetyl-Aluminum and Fluopyram applied 
singly and in combinations and to determine the most 
effective treatment and treatment combinations for 
the management of  Foc TR4 causing Fusarium wilt of  
banana under in vitro test condition.

LITERATURE REVIEW
Fusarium Wilt of  ‘Cavendish’ Banana

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Fusarium wilt, also known as Panama disease have 
threatened banana production worldwide. In the 
Philippines, the disease was suspected to be present in the 
country since the 1970s, but TR4 was only confirmed in 
2008. Its incidence in the surveyed farms increased from 
700 cases in 2005 to 15,000 in 2007 (Molina et al., 2008). 
Large companies currently manage the disease following 
the protocol established for bacterial wilt (Ralstonia 
solanacearum) management, which is based on quarantine, 
sanitation, soil disinfestation and fallows (Molina, 2009). 
To date, Cavendish remains resistant to the Fusarium 
strain that prevails in Central America. However, a 
virulent strain that can attack the Cavendish was found 
causing epidemics in Asia, known as Tropical Race 4 
(TR4). This pathogen destroyed commercial plantations 
of  Cavendish in Taiwan, Northern Territory of  Australia, 
Indonesia, Malaysia, and China making their banana 
exports less competitive. In the Philippines, the disease 
was suspected to be present in the country since the 
1970s, but TR4 was only confirmed in 2008. Its incidence 
in the surveyed farms increased from 700 cases in 2005 to 
15,000 in 2007 (Molina et al., 2008). 
The Pathogen
Fusarium oxysporum f.sp. cubense (Foc) is a soil-borne 
hyphomycete and is one of  more than 100 formae 
speciales of  F. oxysporum that causes vascular wilts of  
flowering plants (Domsch et al. 1980; Nelson et al. 
1983). According to Wardlaw (1961) and Stover (1962), 
the fungus infects the roots of  banana plants, colonizing 
the vascular system of  the rhizome and pseudostem, and 
inducing characteristic wilting symptoms mostly after 
5-6 months of  planting and the symptoms are expressed 
both externally and internally. The fungus produces 
micro- and macroconidia and chlamydospores. Besides, 
it over seasons in infected plants as mycelium and in the 
soil mostly as chlamydospores. The latter survive in the 
soil for at least 20 years (Agrios, 2005). 
The pathogen is broadly categorized into four races, 
namely: races 1, 2, 3 and 4. Races 1, 2 and 4 are pathogenic 
only to banana (Musa spp.) and race 3 is pathogenic 
only to Heliconia spp. All races are present in Australia. 
The pathogen is further categorized into vegetative 
compatibility groups (VCGs) or strains of  which there 
are 21 recognized worldwide. The most important race 
of  this pathogen is race 4, which affects most of  the 
cultivated banana varieties, including ‘Cavendish’ banana 
(Daly and Walduck, 2006).
Symptomatology 
According to Agrios (2005), the symptoms of  Panama 
disease consist of  yellowing of  the oldest leaves or 
lengthwise splitting of  the lower leaf  sheath. Leaves may 
wilt and buckle at their petiole base and, later, younger 
leaves collapse and die. Internally, brown streaks develop 
on and within older leaf  sheaths and these are followed by 
large portions of  the xylem turning brick red to brown. In 
the meantime, the fungus, which enters the banana plant 
from the soil through the feeder roots, advances into the 
xylem vessels of  the rhizome and from there into the 

pseudostem, which it colonizes, resulting in discoloration 
and blockage. 
Whereas, Somrith et al. (2011) reported that the 
symptoms appeared from 5 months in the younger stage 
to the fruit stage. The external symptom is yellowing of  
the leaf  blade and the internal symptom exhibits  reddish 
brown discoloration of  the internal pseudostem. Infected 
plants collapse before maturity. Even though they can 
grow, their yield and the quality of  fruit are decreased.
Disease Cycle
Based on the review of  Pegg, Moore and Bentley (1996), 
Fusarium oxysporum f. sp. cubense is a soil-borne pathogen 
that infects plants through lateral or branching rootlets, 
penetrates the vascular tissue and proliferates in the 
xylem. It restricts movement of  water and induces 
wilting and premature death. The fungus can survive for 
many years in soil as a saprophyte or as chlamydospores. 
Chlamydospores are stimulated to germinate by root 
exudates, particularly those from wounded roots, which 
subsequently become infected.
Microconidia are Sproduced in the xylem within 2-3 days 
on its adaxial side and move upwards in the xylem but 
become trapped at the end walls in the vessels. However, 
hyphae penetrate these end walls and sporulate. Infection 
stimulates the formation of  tyloses and the secretion 
of  gels in the xylem vessels within 24-48 hours, which 
together with trapped microconidia at the perforation 
plates, restrict water movement. Eventually as the plant 
dies, the fungus grows out of  the vascular system into 
the parenchyma after 48-96 hours where it sporulates 
profusely and conidia and chlamydospores are returned 
to the soil (Ploetz (1992).
Disease Epidemiology
The pathogen is spread primarily in infected rhizomes 
(suckers), which are used traditionally for the vegetative 
propagation of  banana. Less frequently, the pathogen 
is spread as spores in soil, running water, and on farm 
equipment and machinery (Agrios, 2005). PCARRD-
DOST (2006) supported those means of  spread and 
added that the natural and initial site of  infection is 
through the root tips of  the plant.
Management
There are no economical methods exposed to eliminate 
Foc TR4 in the soil. Current management practices 
involved implementation of  strict quarantine measures to 
prevent the transfer of  diseased planting materials into 
new areas. Infected banana plants, including those within 
a 6-m radius, must be immediately eradicated to minimize 
the spread of  the disease PCARRD-DOST (2006).
The most effective control can be or achieved by 
planting banana varieties resistant to the existing races 
of  the pathogen. Planting pathogen-free rhizomes in 
pathogen-free soil is also effective. Likewise, the use of  
tissue culture-produced propagative material free of  the 
pathogen is helpful (Agrios, 2005). 
There are reports, however, that maintenance of  an active 
micro-flora in the soil was advocated as a general control 
measure to be used in conjunction with somaclones 

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showing some resistance. Also, various organisms found 
to suppress Foc in laboratory and glasshouse experiments 
have been advocated as biocontrol agents, but little 
success has been reported using strains of  Trichoderma, 
Pseudomonas, Streptomyces, non-pathogenic Fusarium 
of  both rhizospheric and endophytic microbes in the field 
(Zhang et al., 2013). Today, large companies currently 
manage the disease following the protocol established for 
bacterial wilt (Ralstonia solanacearum) management which 
is based on quarantine, sanitation, soil disinfection and 
fallows (Molina, 2009). 
A Chinese research team also reported that crude extract 
of  Chinese chive (Allium tubererosum) inhibited the in 
vitro growth of  Foc ‘tropical race 4’. In field trials in an 
area heavily infested by Foc ‘tropical race 4’, Chinese 
chive in rotation with banana, reduced incidence of  
Fusarium wilt by 88 %-97 % and a disease severity index 
by 91-96 % (Huang et al., 2012). The results of  further 
work indicated that volatiles released from root exudates 
or leached roots and leaves may be responsible (Zhang et 
al., 2013).
Effects of  Fosetyl-Al
Fosetyl-Al is a systemic fungicide which is the breakdown 
product of  Foli-R-Fos that has an active ingredient of  
Phosphonate, the anion of  Phosphonic acid, which is 
used as fungicides against the pathogens belonging to the 
order Peronosporales (Davis et al., 1994). This fungicide 
is known for its direct effect on the fungus that stops 
plant infection by inhibiting spore germination and the 
penetration of  the pathogen into the plant or blocks 
mycelial development and sporulation in the case of  a 
curative treatment and its indirect effect of  slowing down 
significantly the invasion of  the pathogen by reinforcing 
the defensive reactions of  the plants. This enables the 
host’s hypersensitive response to seal-off  the invading 
organism (Bayer Crop Science, 2013).
Fosetyl-al is rapidly absorbed by the plant roots or leaves 
and is translocated in both an upward and downward 
direction, particularly to the growing points. In this 
way, the foliage and root growth developing between 
treatments is protected, including the root hairs of  the 
plants receiving foliar spray application. It also exhibits 
certain level of  curative activity. Best results, particularly 
against the diseases that develop rapidly, are generally 
obtained when treatments commence prior to the onset 
of  infection (Bayer Crop Science, 2012).
Many researches have shown the potential of  this 
fungicide in controlling bacterial and fungal organisms 
belonging to the classes other than Oomycetes. Taylor and 
Washington (1984) reported, fosetyl-al has significantly 
reduced cankers on peach caused by Phytophthora 
cactorum when applied as curative treatments.  On the 
other side, application of  these phosphonates at higher 
concentration was found to provide some control against 
Panama disease of  banana caused by Fusarium oxysporum 
f.sp. cubense (Davis & Bruce, 1996). However, the degree 
of  control varied with the banana growing season and 
from one locality to another. Preliminary investigation in 

the study of  Davis and his co-workers (1994) on Foc in 
culture confirmed that potassium phosphonate inhibited 
mycelial growth, but showed that the degree of  inhibition 
was extremely sensitive to phosphate levels in the growth 
medium and that at concentrations of  phosphate likely 
to be present in plant tissues, phosphonate effects were 
evident only at concentrations in the region of  25 mM. 
On the other hand, Landschoot and Cook (2005) claimed 
that posphonates were not effective as substitute for 
phosphate fertilizers but USDA scientists observed that 
phosphonates have a delayed phosphorus response in 
the plants. Subsequent research revealed that there is a 
conversion of  phosphites into phosphates in the soil with 
the aid primarily of  soil-borne bacteria. This result helps 
in the enhancement of  quality and yield of  the plant.  
Effects of  Bacillus subtilis
The biological control of  plant diseases is an excellent 
alternative strategy for effective control of  various 
diseases as well as augmentation of  nutrient availability in 
the rhizosphere. Several studies have indicated the good 
potentials of  B. subtilis as an effective biological control 
agent of  different plant pathogens (Saha et al., 2012; 
Serafini, 2000; Pukall et al., 2005; Killani et al., 2011, 
Seema and Devaki, 2012; Abd Allah, 2005; Pleban et al., 
1997).   
Bacillus subtilis is an ubiquitous naturally- occurring 
saprophytic bacterium that is commonly recovered from 
soil, water, air, and decomposing plant material. It is a 
biofungicide which was started to be used as a seed-
dressing biofungicide, with registrations in more than 
seven crops (Backmann et al. 1994). Preliminary studies 
conducted by Saha and his comrades (2012) revealed in 
vitro evidences which indicated multiple modes of  action 
of  B. subtilis including antibiosis, effective colonization 
and plant growth promotion which revealed their 
potential for field application and commercial use as 
biocontrol agents.
B. subtilis bacteria produce a class of  lipopeptide antibiotics 
including iturins that help the bacteria out-compete other 
microorganisms by either killing them or reducing their 
growth rate (CPL Scientific Publishing, 2001). In addition, 
Warkentin (2012) and Edgecomb, together with Manker 
(2006), conveyed that this bacterium produces fungicidal 
metabolites such as lipopeptides that would breakdown 
pathogen cell membranes causing the pathogen to 
collapse and die. They also added that this bacterium 
can be used as Plant Growth Promoting Rhizobacterium 
(PGPR) because of  its ability to produce 2,3 butanediol 
which is a volatile compound known to trigger plant 
growth promotion. 
Serafini (2001) reported that B. subtilis inhibits plant 
pathogen spore germination, disrupts germ tube growth, 
and interferes with the attachment of  the pathogen to 
the plant. He further discussed that this bacterium is 
also reported to induce systemic acquired resistance 
(SAR) against bacterial pathogens and boosted the plant’s 
responses against the antagonists.
The production of  antibiotics by the Bacillus spp. and 

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their uses in the biological control of  plant pathogens 
have been reported in many reviews. B. cereus produces 
lytic enzymes and antibiotics; and B. subtilis possesses 
a lytic factor in its cell wall (Pukall et al. 2005). In an 
earlier report, Young et al. (1974) stated that B. subtilis 
produces at least five different antibiotics, namely: 
subtillin, bacitracin, bacillin, subtenolin, and bacilonycin 
that accounts for the inhibition. Moreover, Pukall et al. 
(2005) identified four toxin-producing strains of  Bacillus 
spp., namely B. pumils, B. fusiformis, B. subtilis, and B. 
mojavensis apart from normal toxin producer, B. cereus.
Several researches have reported the production of  
toxins by Bacillus spp. The Bacillus subtilis used in the 
study of  Killani together with his co-workers (2011) 
successfully inhibited the growth of  all the root/soil-
borne fungal pathogens isolated from cowpea in-vitro. 
They described that the production of  antibiotics by 
Bacillus spp. contributes a big factor. Similarly, in the study 
conducted by Seema and Devaki (2012), B. subtilis showed 
50% inhibition of  Rhizoctonia solani in in vitro test. They 
concluded that this may be due to the presence of  the 
antibiotic like inturin A and surfactin produced by B. 
subtilis that has been supported by Akihiro et al. (1993). 
They described that B. subtilis produced antifungal peptide 
antibiotic inturin A and surfactin and further discussed 
that this inturin A has a strong antifungal activity when 
compared to surfactin. 
Moreover, many experiments had established and 
revealed the potential of  this bacterium against pathogens 
causing diseases. For instance, Abd-Allah (2005) reported 
that B. subtilis protected peanut seeds from the negative 
effect of  Sclerotium rolfsii and significantly increased 
growth parameters of  pods compared to the negative 
control. Likewise, application of  a chitinase-producing 
strain of  Bacillus cereus directly to soil significantly 
protected cotton seedlings from root rot disease caused 
by Rhizoctonia solani (Pleban et al., 1997). 
Effects of  Fluopyram
Fluopyram is a succinate dehydrogenase inhibitor (SDHI) 
fungicide. Succinate dehydrogenase (SDH) is an enzyme 
complex couples the oxidation of  succinate to fumarate 
in the mitochondrial matrix (or in the cytoplasmic 
membrane of  bacteria) with the reduction of  ubiquinone 
(UQ), a hydrophobic membrane bound electron carrier, 
to ubiquinol (UQH2) in the membrane during aerobic 
respiration (Horsefield et al., 2006). In addition to its 
function as a dehydrogenase in the respiratory system, 
complex II plays an important role in the tricarboxylic 
acid cycle. The mitochondrial SDH complex is composed 
of  a membrane-peripheral domain and a membrane 
anchor domain (Avenot and Michailides, 2010). 
SDHI fungicides play an important role in plant 
protection against many phyto-pathogenic fungi by 
specifically binding to the ubiquinone- binding site 
(Q-site) of  the mitochondrial complex II, thereby 
inhibiting fungal respiration. These fungicides have been 
used internationally since the late 1960s and are highly 
effective against basidiomycete pathogens such as rusts 

or Rhizoctonia sp and even nematodes. Due to their 
unique mode and site of  action, they show no cross 
resistance with other chemical classes such as strobilurins, 
benzimidazoles or anilinopyrimidines and therefore are 
excellent candidates for managing fungicide resistance 
development and optimizing diseases control (Avenot et 
al., 2008; Leroux et al.,2003; Stammler et al.,2007; Zhang 
et al., 2007). 

MATERIALS AND METHODS
Experimental Design and Treatments 
The study was laid-out in Completely Randomized 
Design (CRD) with eight treatments and replicated three 
times with five plates per replicate or a total of  120 plates.
Treatments used in the study were as follows: T1- Control; 
T2- B. subtilis alone; T3-Fosetyl-Aluminum alone; T4-
Fluopyram alone; T5- B. subtilis + Fosetyl-Aluminum; 
T6-B. subtilis + Fluopyram; T7-Fosetyl-Aluminum + 
Fluopyram and T8-B. subtilis + Fosetyl-Aluminum + 
Fluopyram. Five hundred millilitre of  water solutions 
were prepared for these products with the following 
recommended rates based on their product labels: 5 ml 
of  B. subtilis, 2.14 g of  Fosetyl-Aluminum and 0.50 ml 
of  Fluopyram.
Isolation and Mass Production of  the Pathogen
Isolation protocols for F. oxysporum f.sp. cubense described 
by Vicente et. al (2014) was adopted in this experiment. 
The pure culture of  the pathogen was then mass produced 
in PDA and were incubated for seven days.
Test of  Efficacy of  Different 
Commercial Products against FocTR4
To test the efficacy of  different commercial products 
under laboratory condition, different treatments 
with different concentrations applied singly and in 
combinations was tested against Fusarium oxysporum f. 
sp. cubense (Foc) TR4 using poisoned food technique by 
Burgess et.al (2008). All products were calibrated based 
on their rates per one liter water solution and were added 
to the previously-sterilized medium and shaken prior 
for pour-plating. Pure culture disks of  Fo were obtained 
using sterilized cork borer and were planted at the center 
of  the treated medium. Thereafter, all plates were then 
incubated under room temperature. Observations and 
measurement of  the zones of  growth was done after 
seven days of  incubation.
Validation Test
This was conducted to validate the effect of  each treatment 
either the product causes fungistatic or fungistasis effect. 
This was conducted by transferring the culture disk of  
the pathogen from the treated medium after seven days 
unto the untreated culture medium. All plates were then 
incubated for another seven days for observation.
Data Gathered
Diameter of  Colony
The diameter of  the fungal colony was recorded by taking 
the average of  two long and two short diameters taken at 
right angles for each colony measured in millimetre (mm) 
using a ruler.

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Zone of  Inhibition (ZI)
Zone of  inhibition (ZI) of  the different rates of  
commercial preparation of  B. subtilis was determined 
by measuring the growth of  F. oxysporum f.sp. cubense 
TR4 after 10 days of  incubation and % inhibition was 
calculated using the formula below (Alwathnani and 
Perveen, 2012): 
Inhibition (%) = (C-T/C) 100
Where, C is the average colony diameter in control plate 
and T is the average colony diameter in treated plates. 
Number of  Spores
Spores were counted using a haemacytometer. A four mm 
disc of  FocTR4 were cut from the near center portion 
of  the culture plate using a cork borer and placed in a 
test tube with 10 ml of  sterilized distilled water and were 
shaken well to dislodge the spores. One drop of  this spore 
suspension as placed in a haemacytometer and the spores 
were counted (Ramirez-Peralta et al, 2013). The spores 
were counted after the termination on the laboratory 
test. Basic procedure of  using haemacytometer in the 
counting of  spores was followed using the formula of  
Quimio and Quimio (2001):
Number of  Spores = A+B+C+D+E
(50, 000 for microspores and 2,000 for macrospores)
Validation Test
Results of  the validation test will be based on the growth 
of  the pathogen. If  the transferred culture disk of  the 
pathogen from the treated plate grew (+), this means that 
the product only causes fungistasis effect or just inhibited 
the growth of  the pathogen. Whereas, if  the disk does 
not grew (-), then the product causes fungistatic effect or 
totally killed the pathogen.               
Statistical Analysis
The study was analysed using Analysis of  Variance 
(ANOVA) in Completely Randomized Design and the 
differences among treatment means was compared using 
Tukey’s Honest Significant Difference (THSD).

RESULTS AND DISCUSSION
Inhibitory Effects of  Different Commercial Products 
Applied Singly and in Combinations Against FocTR4 

Treatment effects of  different commercial products 
applied singly and in combinations against FocTR4 is 
presented in Table 1. Inhibitory effects of  the products 
on the morphological characteristics of  FocTR4 and 
validation of  their efficacy is shown in Table 2. Analysis 
of  Variance revealed significant differences among 
treatment means in all parameters. 
Results showed that treatments of  Fosetyl-Al alone 
and the combinations of  the three products, B. subtilis, 
Fosetyl-Al and Fluopyram had totally inhibited (100.00%) 
the growth of  FocTR4 after 10 days of  observation. 
Results also showed that single treatment of  B. subtilis 
had significantly inhibited (51.03%) FocTR4 with colony 
diameter of  39.38mm. Single treatment of  Fluopyram, 
on the other hand, showed lower inhibition (12.78%) 
with 70.10mm FocTR4 colony diameter. Fosetyl-al alone 
and the combinations of  the three products products 
also inhibited the production of  FocTR4 spores with 
zero micro- and macrospores. Comparably, lower 
macrospores production of  FocTR4 was obtained by 
the plates with single treatment of  Fluopyram and in the 
control plate with average number of  spores of  1,333.00 
and 5,000.00 which subsequently produces 11,983,333.33 
and 25,983,333.33 microspores. Highest number of  
FocTR4 population count was observed in plates treated 
with B. subtilis alone with micro- and macrospores of  58, 
866, 666.67 and 43,333.00, respectively. 
Results of  the validation test of  the efficacy of  the 
products (Table 1& 2) showed that the inhibition of  
single treatments of  these products and the combination 
of  Fosetyl-Al and Fluopyram was mainly due to its 
fungistasis effect which only inhibit the growth of  the 
fungus as indicated by the positive symbols (+). In the 
case of  Fosetyl Aluminum, presence of  chlamydospores 
(survival structure) were clearly observed which might 
implies the possibilities of  the product to induce 
chlamydospore production. On the other hand, treatment 
of  B. subtilis combined with Fosetyl-Al or Fluopyram and 
the combinations of  the three products showed that their 
combinations can cause fungistatic effect which totally 
kills the fungus as indicated by the negative symbol 

Table 1. Effects of  single and combined treatments of  different commercial products on the colony diametera,  
percent growth inhibitionb and spore population count per millilitrec of  FocTR4 after 10 days of  incubation and 
validationd of  their effects toward the pathogen.

Treatments
Colony 
Diameter 
(Mm)
**

% Zone Of  
Inhibition
**

Foc Tr4 Spore Population Count/
Millilitre Validation Test
Micro** Macro**

Control (SDW only) 80.41d 0.00d 25,983,333.33c 5,000.00a (+)
B. subtilis 39.38b 51.03b 58,866,666.67d 43,333.00b (+)
Fosetyl-Aluminum 0.00a 100.00a 0.00a 0.00a (+)
Fluopyram 70.10c 12.78c 11,983,333.33b 1,333.00a (+)
B.subtilis + Fosetyl-Al 0.00a 100.00a 0.00a 0.00a (-)
B.subtilis +Fluopyram 0.00a 100.00a 0.00a 0.00a (-)
Fosetyl-Al + Fluopyram 0.00a 100.00a 0.00a 0.00a (+)
B. subtilis + Fosetyl-Al + 
Fluopyram

0.00a 100.00a 0.00a 0.00a (-)

CV (%) = 2.92 0.67 5.21 19.45

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Table 2. Inhibitory effects of  B. subtilis, Fosetyl-Al and Fluopyram applied singly and in combinations against  t h e 
morphological characteristics of  Foc TR4. A) Colony growth of  Foc TR4 after 10 days of  observation; B) Effects on 
the hyphal growth and structures of  Foc TR4 taken from the Foc-inoculated agar block in the treated medium; C) Foc 
TR4 spore population per millilitre taken from the near center of  he treated medium; D) Growth of  Foc TR4 in fresh 
and untreated medium after transfer from treated medium to validate their effects toward the pathogen.
Treatments Colony Growth Hyphal Growth Spore Population Validation Test

A B C D
Control (SDW only)

B. subtilis alone

Fosetyl-Aluminum 
alone

Floupyram alone

B.subtilis+ Fosetyl-Al

B.subtilis + Fluopyram

Fosetyl-Al+ 
Fluopyram

B. subtilis + Fosetyl-Al 
+ Fluopyram

(-). Data were replicated thrice with three plates per 
replication. 
Means having the same superscript means there were no 
significant differences at 1% level, THSD.
a = Colony diameter (mm) was measured by computing 
the average of  the two longest and two shortest growth  
diameters in plates.
b = Percent of  zone of  inhibition was computed using 
the formula: C-T/C X 100, where, C is the average 
colony  diameter in control plate and T is the average 
colony diameter in treated plates. 
Data were subjected to arc-sine transformation prior to 
analysis.
c = Foc TR4population count per millilitre was computed 
using the formula: (A+B+C+D+E) times 50,000 
(for microspores) and 2,000 (for macrospores).
d=Validation test was conducted by transferring the agar 
disc from the treated plates onto a fresh medium to test the 
efficacy of  the products on and after treatment application: 
(+) means that FocTR4 grow on the fresh medium after 

transfer.  Using of  Fosetyl-Al against FocTR4 can totally 
inhibit the hyphal growth. The results may be attributed 
by active ingredient of  fungicide. Where in, fosetyl-Al 
and other salts of  phosphorous acid has a break down 
product of  Phosphite ion, which is the mainactive 
ingredient against Oomycete pathogens (Alviter, 2006). 
Investigation on the Phytophthora sp. concluded that 
phosphite interferes with key phosphorylating enzymes 
and phosphorous metabolism in general, affecting 
the synthesis of  different phosphorous-containing 
compounds essential for its growth and development such 
as Nicotinamide adenine dinucleotide (NAD), Adenosine 
triphosphate (ATP) and nucleotides. Moreover, Fosetyl-Al 
possess excellent systemic activity against several diseases 
caused by Phytophthora sp. and can inhibit mycelial 
growth and sporulation of  the fungus (Boughalleb, 
2006).  Khaskheli and his comrades (2017) reported that 
the mycelial growth of  Fusarium nivale causing mango 
malformation disease was significantly inhibited by 
Fosetyl- Aluminium in their in vitro study.  Futhermore, 

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Alviter (2006) discussed that the effects of  phosphite on 
Phytophthora sp. grown at low level phosphates shown to be 
greater, suggesting an antagonistic relationship between 
phosphite and phosphate under in vitro conditions. The 
effect of  Fosetyl-al on the structures of  FocTR4 induces 
chlamydospore (Figure 2B). 
However, in the spore population count, plates treated 
with B. subtilis showed highest number of  micro and 
macrospores as compared to other treatments. Also, plates 
treated with fosetyl-aluminum has no spore formation. 
These cases might be correlated to the concept of  the 
second hypothesis of  compensation on which a specific 
“weakness” can be compensated by a specific “strength” 
(Bashi and Rotem, 1974, 1975a and Bashi et al, 1973). 
In order to compensate the activities of  B. subtilis 
on degrading the cell wall of  the hyphal structure of  
FocTR4, the pathogen produced more spores for it to 
compensate the degradation of  its hyphae and in order 
for it to survive. 
The same goes with the reaction of  FocTR4 to the 
effect of  fosetyl-Al on which it induced itself  to produce 
chlamydospores for it to compensate the effect of  the 
product against its spores as previously discussed, 
fosetyl-al targeted mainly its spore formation by affecting 
the synthesis of  different phosphorous-containing 
compounds essential for its growth and development.
On the other hand, Fluopyram is a succinate 
dehydrogenase inhibitor (SDHI) fungicide. Succinate 
dehydrogenase (SDH) is an enzyme complex couples the 
oxidation of  succinate to fumarate in the mitochondrial 
matrix (or in the cytoplasmic membrane of  bacteria) 
with the reduction of  ubiquinone (UQ), a hydrophobic 
membrane bound electron carrier, to ubiquinol (UQH2) 
in the membrane during aerobic respiration (Horsefield et 
al., 2006). In addition to its function as a dehydrogenase 
in the respiratory system, complex II plays an important 
role in the tricarboxylic acid cycle (Kreb’s Cycle). 
The mitochondrial SDH complex is composed of  a 
membrane-peripheral domain and a membrane anchor 
domain (Avenot and Michailides, 2010). Nevertheless, 
SDHIs are classified as medium to high risk for resistance 
development because of  their single-site mode of  action.   

CONCLUSION
Based on the results, generally, it can be concluded that 
Fosetyl-Al alone and its combination to B. subtilis or 
Fluopyram and the combination of  the three fungicides 
can potentially mitigate Foc TR4 population which 
causes the most devastating disease of  banana. However, 
further validation of  their effects under pot and field 
tests should be undergone. Moreover, the combination 
of  these products to other available strategies to improve 
its efficacy can be explored in the future.

Acknowledgement
Authors would like to acknowledge University of  
Southeastern Philippines-Tagum-Mabini Campus for 
allowing the conduct of  the experiment and the utilization 

of  lab materials.

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