Journal of Natural Pesticide Research 6 (2023) 100051

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Journal of Natural Pesticide Research

journal homepage: www.journals.elsevier.com/journal-of-natural-pesticide-research

Efficacy of microbial antagonists in the management of bacterial wilt of

field-grown tomato ]]
]]]]]]
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Arthur O. Wamania,b, , James W. Muthomia, Eunice Mutitua, Wanjohi J. Wacekeb

⁎

a
Department of Plant Science and Crop Protection, University of Nairobi, P. O. BOX 30197, 00100 GPO, Nairobi, Kenya
b
Department of Agriculture Science and Technology, Kenyatta University, P.O. BOX 43844, 00100, Nairobi, Kenya

A R T I C L E I N F O A B S T R A C T

Keywords: Bacterial wilt caused by Ralstonia solanacearum can cause up to 100% yield loss in tomato production and has
Biological control compelled many farmers to abandon previous productive farms. Consequently, the absence of an effective
Trichoderma spp. control method demands an intensive such for a functional management option. The study was carried out to
Microbial biopesticides evaluate the efficacy of microbial antagonists in managing bacterial wilt under field conditions. The experiment
Bacterial wilt
was laid down under Randomized Complete Block Design and four replicates maintained for each treatment.
Tomato
Antagonistic strains of locally isolated Trichoderma hamatum, T. atroviride, T. harzianum, Bacillus subtilis, Serratia
spp., and Acinetobacter spp. were assessed. Commercial formulations of T. viride (Bio Cure F®) and Pseudomonas
fluorescence (Bio Cure B®) were included as standard checks. Treatment application was initiated at trans­
planting by drenching the rhizosphere with 50 ml of the inoculum. Application was repeated every two weeks
until tenth week after transplanting. Data was collected on plant stand count, disease incidence, disease severity,
plant height, plant biomass, and fruit weight. Trichoderma hamatum was the most effective in experiment 1,
reducing crop mortality, incidence and AUDPC by up to 51.7%, 49.3% and 58.2%, respectively. It also exhibited
the highest percentage yield increase by up to 196.4% compared to the untreated control. In contrast, B. subtilis
showed superior disease suppression in experiment 2, reducing crop mortality, incidence and AUDPC by up to
44.6%, 48.5% and 51.0%, respectively. It also increased biomass by approximately 62.0% in both experiments.
It was closely followed by Serratia spp., which also gave the highest yield output in experiment 2 of up to
233.0%. These findings suggest that novel antagonistic strains T. hamatum and B. subtilis presents unique op­
portunities for exploiting local microbial resources as biocontrol products. These products can be incorporated as
part of an integrated bacterial wilt management program in farms where production is significantly affected by
the disease.

1. Introduction 90% of farms in Embu County (Kago et al., 2016). Kenyan farmers cited
this disease as the most problematic tomato disease to manage (Kago
Bacterial wilt is a highly destructive plant disease with a broad host et al., 2016; Sharma et al., 2022) and had been previously reported as
range and wide geographical distribution (Hayward, 1991; Kaguong’o the most damaging disease, hindering the production of solanaceous
et al., 2010; Abdurahman et al., 2019). This disease infects at least 450 crops in the major production regions in the country (Kaguong’o et al.,
plant species in over 54 taxonomic families (Kurabachew and Ayana, 2010; Sharma et al., 2022). Consequently, many farmers have been
2016; Lavale et al., 2022; Kansal et al., 2023). The causative pathogen, forced to abandon production in formerly productive greenhouses and
Ralstonia solanacearum, is a species complex with multiple genetic fields once the pathogen is established (Hayward, 1991; Government of
variabilities at species levels (Meng, 2013; Abdurahman et al., 2019; Kenya, 2015; Aloyce et al., 2017).
Lavale et al., 2022; Sharma et al., 2022). It is an endemic pathogen and To date, no single, economically feasible method has offered com­
can survive in the soil for long periods in the absence of an ideal host plete protection against infection and subsequent yield losses from
plant (Hayward, 1991; Kaguong’o et al., 2010; Lavale et al., 2022). bacterial wilt (Njau et al., 2021). The wide host range and survival
In Kenya, the prevalence of bacterial wilt was reported in 77% of longevity of the pathogen in the soil render crop rotation less effective
farms surveyed in 10 former districts (Kaguong’o et al., 2010) and in (Kaguong’o et al., 2008; Chakravarty, 2023). Several chemical

⁎
Corresponding author at: Department of Plant Science and Crop Protection, University of Nairobi, P. O. BOX 30197, 00100 GPO, Nairobi, Kenya.
E-mail address: arthurwamani@gmail.com (A.O. Wamani).

https://doi.org/10.1016/j.napere.2023.100051
Received 7 December 2022; Received in revised form 18 April 2023; Accepted 3 July 2023
2773-0786/© 2023 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/
by-nc-nd/4.0/).
A.O. Wamani, J.W. Muthomi, E. Mutitu et al. Journal of Natural Pesticide Research 6 (2023) 100051

fumigants such as metam sodium, chloropicrin and 1,3-di­ 121 °C for 30 min. After cooling, the sorghum was inoculated with five
chloropropene, bactericides such as streptomycin sulfate and resistance mycelial agar plugs of each antagonist cut from the edges of seven-day-
activators such as acibenzolar-s-methyl have been reported to effec­ old cultures using a 5 mm diameter cork borer. The polythene bags
tively reduce bacterial wilt incidence and increase the yield of field- were incubated at room temperature (24 ± 2˚C) for 21 days. The bags
grown tomato (Yuliar and Toyota, 2015). However, their use has been were periodically opened under sterile conditions to facilitate aeration
highly discouraged due to associated environmental drawbacks and and then briefly shaken for dispersal of spores and mycelia. The colo­
high levels of resistance development (Njau et al., 2021). nized sorghum was aseptically air-dried at 27 °C, ground with a blender
Breeding has produced some tomato varieties with moderate to and mixed with sterilized talcum powder in the ratio of 1:1 w/w, then
strong resistance against bacterial wilt. However, even the most re­ thoroughly homogenized.
sistant varieties also exhibit latent infection (Michael et al., 2020; The concentration of the colony forming units (CFU) of the for­
Oussou et al., 2020). Some of these resistant varieties also exhibited mulation was determined by serial dilution and pour plate technique
inferior fruit qualities (Yuliar and Toyota, 2015). On the other hand, with subsequent colony counts (Niranjana et al., 2009). The formula­
most of the resistant varieties with desirable fruit qualities are hybrids tion was further diluted with sterile distilled water (SDW) to achieve
and their seeds are too expensive for the majority of small-scale farmers the desired population of 1 × 107CFU/ml for field experiments.
(Zohoungbogbo et al., 2021). The alternative approach of grafting with
resistant rootstock also has cost implications that hinder most small- 2.3. Multiplication and formulation of bacterial antagonists
scale farmers' adoption of the technology (Manickam et al., 2021).
Consequently, the majority of farmers in Kenya (79%) rogue infected Bacterial antagonists were mass cultured on nutrient broth and then
plants as a last resort in an effort to minimize yield losses (Okello et al., mixed with talcum powder following a slight modification of the pro­
2020). cedure described by Martínez-Álvarez et al. (2016). Purified distinct
Not all hope is lost though, the incorporation of biological control colonies were picked from a 48-hour antagonistic bacterial isolate
products in the management of pests and diseases of crop plants has culture and aseptically inoculated in 100 ml of sterile nutrient broth
delivered some promising results (Senthil-Nathan, 2014). Microbial contained in a 250 ml conical flask. Subsequently, the broth was in­
biocontrol products offer a promising alternative for the management cubated at room temperature (24 ± 2˚C) for 72 h in a shaker set at
of bacterial wilt (Jiang et al., 2017). They are safer, ecofriendly, pro­ 150 rpm. Following the successful growth and multiplication of an­
ductively sustainable and possess a minimal risk for resistance build-up tagonists, the broth was mixed with sterile talcum powder in the ratio
(Kohl et al., 2019). Further, their efficacy is comparable to that of of 1:2 v/w. The talcum powder had previously been mixed with car­
chemical pesticides (O’Brien, 2017). Even though considerable success boxyl-methyl-cellulose (1% w/w), and calcium carbonate (15% w/w)
in the management of bacterial wilt of field-grown tomatoes using lo­ and then autoclaved at 121˚C, 15 PSI for 30 min before use. The mixture
cally isolated microorganisms was recently reported in Kenya (Kariuki was aseptically dried in an oven at 28 °C for five days, then ground with
et al., 2020), the potential of local microbial resources in managing this a blender to homogenize and stored at 4 °C until use.
disease under field conditions is yet to be fully exploited. Subsequently, The cell density of the formulation was determined through colony
the current study was conducted to investigate the biocontrol potency counts. Colony count plates were prepared through spread-plating
of locally isolated antagonistic bacteria and fungi against bacterial wilt 100 µL aliquots of serial dilutes from 108 to 1012 dilutions on pre-dried
on field-grown tomatoes in Kenya. surfaces of nutrient agar. The formulation was further diluted with SDW
to obtain a cell density of 1 × 108 CFU/ml desirable for the field ex­
2. Materials and methods periment.

2.1. Description of the study area 2.4. Field experiment layout and design

The field experiment was carried out in a farmer’s field that had The field experiment was carried out following the experimental
been naturally infected with bacterial wilt in Kirinyaga County at procedure described by Morsy et al. (2009). The experiment was car­
geographical coordinates 0°44.8765’S, 37°24.8421’E. The experimental ried out on a farmer’s field that had been naturally infected with bac­
site is located in an agro-ecological zone (AEZ) Lower Midland (LM) 4, terial wilt. The field had a history of repeated tomato cultivation and
between 1090 m and 1220 m above sea level. The region receives mean had recorded 50% bacterial wilt incidence in the previous cropping
annual rainfall ranging from 800 to 950 mm distributed in two seasons. season. The experiment was set in a Randomized Complete Block De­
The first rainy season starts at the end of March and produces an sign (RCBD) with four replicates for each treatment. Experimental plots
average of 400 mm while the second rainy season commences in mid to measured 3 × 3 m with a one-meter spacing between adjacent plots.
end of October through to January with an average of 300 mm per The first experiment was carried out between July and October 2018
season. The temperature ranges from 13.7° to 30.4°C with an annual and a repeat experiment between December 2018 and April 2019.
mean of 22 °C (Jaetzold et al., 2010). However, the daily rainfall and Furrow irrigation was used in both experiments in accordance to crop
average, maximum and minimum temperatures considerably varied moisture needs.
over the experiments’ timeline (Fig. 1). Three weeks old Rio Grande variety tomato seedlings raised in a
The soil at the experimental site was texturally classified as clay soil nursery were transplanted in holes dug at a spacing of 60 × 60 cm. The
and had high proportions of clay (58%), moderate proportions of sand holes had previously been incorporated with Di Ammonium Phosphate
(30%) and low proportions of silt (12%). The soil had a pH of 7.3, low (DAP) fertilizer at the rate of seven grams per hole, accumulating to
levels of total nitrogen (0.15%), and a total organic carbon percentage approximately 194 kg/Ha. Top dressing with CAN was done at four and
of 1.54, giving a C/N ratio of 10.27. The concentration of other ele­ eight weeks after transplanting at the rate of four and eight grams per
ments was as indicated in Table 1. plant, accumulating to approximately 111 and 222 kg/Ha, respectively.
Agronomic practices and watering were implemented appropriately.
2.2. Multiplication and formulation of fungal antagonists Trichoderma hamatum, T. atroviride, T. harzianum, Bacillus subtilis,
Serratia spp. and Acinetobacter spp. were assessed for biocontrol po­
Multiplication of fungal antagonists on sorghum was carried out tency. Standard checks including commercial T. viride (Bio cure F®),
following a slight modification of the method described by Mishra et al. commercial Pseudomonas fluorescence (Bio cure B®), and negative
(2011) and Kariuki et al. (2020). Briefly, 200 g of sorghum was soaked control were also included in the experiment. Treatment application
in water for 12 h and autoclaved in heat-resistant polythene bags at was initiated at transplanting by drenching individual plants with 50 ml

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A.O. Wamani, J.W. Muthomi, E. Mutitu et al. Journal of Natural Pesticide Research 6 (2023) 100051

Fig. 1. Daily rainfall and temperature recorded data over the project implementation period inexperiment 1 (a) and experiment 2 (b), respectively.

Table 1 formula; (Number of dead plants per plot/total number of plants per
Concentration of mineral elements in soils collected from plot) × 100.
the experimental site. The biocontrol efficacy of each antagonist was computed based on
Soil properties Value
their potency in reducing disease incidence, crop mortality, and
AUDPC. The formula by Zheng et al. (2020) was used as follows:
Phosphorus (Olsen) ppm 22.00
Potassium me% 0.40 Biocontrol efficacy
Calcium me% 43.20 DI/CM/AUDPC in control – DI/CM/AUDPC in treated plots
Magnesium me% 4.80 = × 100
Manganese me% 0.40
DI/CM/AUDPC in control
Copper ppm 2.00
Where DI = Disease incidence, CM = Crop mortality and AUDPC
Iron ppm 20.20
Zinc ppm 7.50 = Area under disease progress curve. The computation for Biocontrol
Sodium me% 1.06 efficacy was done using each disease measurement separately.

2.6. Assessment of crop growth and yield attributes

of the inoculum around the root zone. A similar quantity of water was
used for negative control plots. The treatment was repeated every two
Data on plant height and shoot biomass were collected at the
weeks until the tenth week after transplanting (WAT).
flowering stage using the meter rule and digital weighing balance, re­
spectively. For plant height, five plants were randomly sampled in each
2.5. Assessment of bacterial wilt incidence and severity plot from the central row and measured from the soil level to the
terminal growth point. Similarly, for biomass determination, three
Data on disease incidence, severity and crop mortality was recorded plants were sampled from the central row, carefully uprooted, and
from the third week to the tenth WAT. Disease incidence was de­ dried to constant weight in an oven set at 60 °C. Fruit harvesting was
termined by counting the number of plants exhibiting typical bacterial initiated following an exhibition of orange to pink tinges. The cumu­
wilt symptoms in a plot and percentages computed using the formula; lative weight of all marketable fruits was recorded for each plot and
(Number of plants exhibiting wilt symptoms per plot/total number of converted into tons per hectare as described by Suchoff et al. (2019).
plants per plot) × 100 as described by Guo et al. (2004) and Kariuki
et al. (2020). Data on bacterial wilt incidence was used to compute the 2.7. Data analysis
area under the disease progress curve (AUDPC) as previously described
by Shaner and Finney, 1977 and Okumu et al. (2018) using the formula; Data on disease incidence, severity, crop mortality, AUDPC, plant
n 1 height, shoot biomass and fruit yield were processed in Microsoft Excel
[Yi + Yi + 1][Xi + 1 Xi]
AUDPC = 1=n and then subjected to Analysis of Variance (ANOVA) to determine
2 significant differences among the treatments. The means were sepa­
Where Y = incidence (per unit) at the ith observation, Xi = time rated by Fisher’s protected least significant difference at a 5% level of
(weeks) at the ith observation, and n = total number of observations. significance using GENSTAT® 15th Edition. Further, means were sub­
Disease severity was computed by averaging the severity scores jected to Pearson correlation using SPSS version 28.0.
recorded for a given plot. A scale range of 0–4 was used to score se­
verity; where 0 was considered for plants with no visible wilt symptom, 3. Results
1 for plants with up to 25% of their foliage wilted, 2 for plants with
foliage wilting between 26% and 50%; 3 for plants with foliage wilting 3.1. Effect of microbial antagonists on bacterial wilt of tomato
between 51% and 75% and 4 for plants with foliage wilting greater than
75% (Yang et al., 2012; Zheng et al., 2020). Plant stand was determined Incorporation of the biocontrol agents for field management of
by counting the number of surviving plants in a whole plot (Okumu bacterial wilt had significant (P ≤ 0.05) effects on disease incidence,
et al., 2018). The percentage crop mortality was computed using the severity, crop mortality and area under disease progress curve

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A.O. Wamani, J.W. Muthomi, E. Mutitu et al. Journal of Natural Pesticide Research 6 (2023) 100051

Table 2
Percentage disease incidence of tomato plants drenched with different microbial antagonists in field experiments 1 and 2.

Experiment 1 Experiment 2

Weeks after transplanting

Treatments 4 6 8 10 4 6 8 10
T. hamatum 6.7d 16.1c 32.2d 41.1e 8.3d 17.4c 40.2 cd 49.3 cd
T. atroviride 17.0ab 32.2ab 40.0 cd 51.1cde 20.4a 35.6ab 59.9ab 68.5abc
T. harzianum 17.0ab 38.2a 62.2ab 72.2ab 18.3abc 39.3ab 56.9abc 71.3ab
T. viride 13.7abc 33.3ab 50.7bc 62.2bc 21.0a 27.4bc 36.3d 49.2 cd
B. subtilis 9.2 cd 22.2bc 39.9 cd 46.7de 12.4abcd 20.4c 30.2d 41.7d
Serratia spp. 11.1bcd 23.3bc 38.9 cd 51.1cde 10.0 cd 16.4c 34.0d 43.6d
Acinetobacter spp. 15.1abc 30.4ab 48.9bc 60.0c 19.1abc 26.1bc 40.3 cd 55.4bcd
P. flourescens 15.0abc 28.7abc 42.6 cd 53.3 cd 11.2bcd 26.4bc 45.4bcd 57.5bcd
Control 19.0a 41.1a 71.6a 81.1a 19.1ab 45.2a 65.8a 80.9a
Means 13.8 29.5 47.4 57.7 15.5 28.2 45.4 57.5
LSD 56.4 13.4 16.3 11.5 9.1 14.0 18.4 20.0
CV% 26.8 26.1 20.1 11.5 40.2 34.1 27.3 23.8

† Values with the same lower-case letters in a column within the Sub spanner heading are not significantly different at P < 0.05 ‡LSD: Least Significance Difference
§CV%: Coefficient of Variation

(AUDPC). In experiment 1, the highest biocontrol efficacy was observed Similarly, there was a significant (P ≤ 0.05) difference among the
in plots treated with T. hamatum, in which bacterial wilt incidence and biocontrol agents for AUDPC (Fig. 3). The AUDPC ranged from 1083.4
crop mortality were reduced by up to 49.3% and 51.7% respectively, to 2592.4 in experiment 1 and 1289.1 to 2514.1 in experiment 2. The
compared to untreated control. However, in experiment 2, the highest disease intensity between the two experiments did not differ sig­
biocontrol efficacy was observed in plots treated with B. subtilis, which nificantly as experiment 1 had an AUDPC of 15, 715.8 while experiment
exhibited a reduction of up to 48.5% in bacterial wilt incidence. The 2 had 15, 496.9. The highest reduction in AUDPC of up to 58.2% and
highest reduction in crop mortality (38.8%) at 10 WAT was observed in 51.9% was observed in plots treated with T. hamatum and Serratia spp.,
plots treated with Serratia spp. and T. viride (Bio Cure F®) (Fig. 2; in experiments 1 and 2, respectively, compared to untreated control. In
Table 2). both experiments, B. subtilis followed closely in the reduction of AUDPC

Fig. 2. Percentage mortality of tomato plants drenched with antagonistic fungi (a, b) and antagonistic bacteria (c, d) in experiments 1 and 2 respectively.

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A.O. Wamani, J.W. Muthomi, E. Mutitu et al. Journal of Natural Pesticide Research 6 (2023) 100051

Fig. 3. Area under disease progress curve of bacterial wilt on tomato plants drenched with different microbial antagonists for field experiments 1 and 2.

while the least reduction in AUDPC was observed in plots treated with management of bacterial wilt. Albeit the lack of significant
T. harzianum and T. atroviride in experiments 1 and 2, respectively. (P ≤ 0.05) difference in heights of tomato plants treated with dif­
Overall, the results of locally isolated BCAs were comparable, if not ferent biocontrol products, the highest height enhancement was
superior (T. hamatum, B. subtilis and Serratia spp.) to the biocontrol potency observed in plots treated with T. viride in experiment 1 while plots
of commercial standard checks. There was a conspicuous fluctuation in the treated with Serratia spp. also exhibited superior height enhance­
biocontrol efficacy of the BCAs between experiments 1 and 2 at ten WAT. ment in experiment 2 (Table 4).
The effectiveness of Acinetobacter spp., B. subtilis and Serratia spp. in­ Plant biomass was significantly (P ≤ 0.05) different in both ex­
creased by 5.5%, 6.1% and 9.1%, respectively, in reducing wilt incidence periments. Overall, plots treated with bacterial antagonists recorded
between experiments 1 and 2. In contrast, the efficacy of P. fluorescens (Bio higher biomass compared to plots treated with fungal antagonists. The
cure B®) decreased by 5.4%. In the case of fungal BCAs, an increase in highest plant biomass was recorded in plots treated with B. subtilis,
biocontrol efficacy of up to 15.9% was exhibited by T. viride (Bio Cure correspondingly exhibiting an increment of 62.0% and 62.3% in ex­
F®). Trichoderma harzianum also showed a negligible increase of only periments 1 and 2, respectively, compared to untreated control.
0.9%. However, a reduction of 10.3% and 21.7% was exhibited by T. However, plots treated with T. harzianum had 2.2% and 0.9% less
hamatum and T. atroviride, respectively (Table 2). biomass in experiments 1 and 2, respectively, compared to untreated
Bacterial wilt severity of tomato plants treated with different an­ control (Table 4).
tagonistic isolates changed between low and high levels over the project Equally, the incorporation of different biocontrol agents resulted in
period despite evidence of significant (P ≤ 0.05) differences between significant (P ≤ 0.05) differences in fruit weight. The highest yield in­
means of treatments. The observed change was a result of rapid bac­ crement of up to 196.4% and 233.1% was recorded in plots treated with
terial wilt development and subsequent plant mortality (Table 3). T. hamatum and Serratia spp. in experiments 1 and 2, respectively,
compared to untreated control. In experiment 1, fruit yield in plots
treated with T. viride and B. subtilis were comparable to yield output
3.2. Effects of microbial antagonists on growth and yield attributes of recorded in plots treated with T. hamatum while in experiment 2,
tomato comparable results were observed between plots treated with Serratia
spp. to those treated with B. subtilis, T. viride, T. hamatum, and
The growth and yield parameters of tomatoes were significantly Acinetobacter spp (Table 4).
affected by the field incorporation of biocontrol agents for the

Table 3
Mean severity of bacterial wilt on tomato plants drenched with different microbial antagonists in field experiments 1 and 2.

Experiment 1 Experiment 2

Weeks after transplanting

Treatments 4 6 8 10 4 6 8 10

T. hamatum 1.7c 3.6ab 2.7 cd 2.8c 1.8bc 2.8ab 3.1a 1.7d

T. atroviride 2.5b 2.0d 1.6e 2.5 cd 3.4a 2.3bcd 2.8a 2.7abc
T. harzianum 3.4a 2.5 cd 2.5 cd 2.8c 2.3bc 3.0ab 3.1a 3.4ab
T. viride 2.8ab 2.7 cd 2.9c 3.0bc 2.0bc 3.1a 2.3a 3.5a
B. subtilis 1.5c 2.9bc 3.0bc 3.4ab 1.5c 1.5d 2.5a 2.0 cd
Serratia spp. 2.8ab 3.4ab 3.5ab 2.8c 2.1bc 1.7 cd 2.2a 2.3 cd
Acinetobacter spp. 1.2c 1.3e 2.8c 2.2d 2.8ab 1.9 cd 2.9a 2.5bcd
P. flourescens 3.2ab 3.9a 2.2de 3.1abc 1.7c 2.3abc 2.7a 2.3 cd
Control 2.9ab 3.0bc 3.6a 3.6a 2.1bc 0.0e 3.3a 2.9abc
Means 2.5 2.8 2.7 2.9 2.2 2.1 2.8 2.6
LSD 0.7 0.7 0.6 0.6 1.0 0.8 0.9 1.0
CV% 15.8 13.9 12.6 11.5 32.3 27 22.4 25.8

† Values with the same lower-case letters in a column within the Sub spanner heading are not significantly different at P < 0.05
‡LSD: Least Significance Difference
§CV%: Coefficient of Variation

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A.O. Wamani, J.W. Muthomi, E. Mutitu et al. Journal of Natural Pesticide Research 6 (2023) 100051

Table 4
Means of height, biomass, and fruit weight of tomato plants drenched with microbial antagonists in field experiments 1 and 2.

Experiment 1 Experiment 2

Treatments Plant height in Dry weight in Fruit weight in Kg/hac Plant height in Dry weight in Fruit weight in Kgs/
Cms grams Cms grams hac.

T. hamatum 40.2 a 33.3ab 2297.0a 37.2 a 35.7a 1553.0a

T. atroviride 39.2 a 28.2c 1756.0bc 35.8 a 27.0bc 769.0bc
T. harzianum 39.8 a 25.6c 1317.0d 36.0 a 23.3c 567.0c
T. viride 41.8 a 32.3b 2128.0ab 38.1 a 32.7ab 1578.0a
B. subtilis 38.9 a 37.1a 2011.0abc 37.8 a 38.3a 1586.0a
Serratia spp. 39.1 a 32.5b 1858.0bc 38.1 a 32.0ab 1602.0a
Acinetobacter spp. 37.2 a 34.8ab 1606.0 cd 36.5 a 34.9a 1369.0ab
P. fluorescens 37.7 a 34.4ab 1203.0de 35.6 a 33.9a 828.0bc
Control 37.1 a 26.2c 775.0e 35.8 a 23.6c 481.0c
Means 38.9 31.6 1661 36.8 31.3 1148
LSD 3.8 4.1 438.1 4.9 6.5 616.2
CV% 6.6 8.9 18.1 9.1 14.2 36.8

†Values with the same lowercase letters in a row within a column are not significantly different at P < 0.05.
‡LSD: Least Significance Difference
§CV%: Coefficient of Variation

3.3. Relationship between bacterial wilt incidence, crop mortality, severity, experiments. There were variations in the biocontrol efficacy of the
plant height and yield attributes different BCAs within and between the two experiments. These results
are in agreement with the findings of Guo et al. (2004), Subedi et al.
The results of correlation analysis exhibited the existence of high (2019), and Kariuki et al. (2020) which also reported variations in
levels of association between different bacterial wilt parameters, plant biocontrol potency of antagonistic isolates in the management of bac­
growth and tomato yield attributes. Among the disease parameters, terial wilt.
crop mortality exhibited significantly strong positive correlation to in­ Bacillus subtilis maintained high biocontrol efficacy in both ex­
cidence and AUDPC in experiments 1 (r = 0.96, p<0.001; r = 0.93, periments even though Serratia spp. exhibited a superior reduction in
p<0.001) and experiment 2 (r = 0.93, p<0.001; r = 0.94, p<0.001), crop mortality and AUDPC in experiment 2. Comparable results on
respectively. In contrast, crop mortality, disease incidence, severity and superior biocontrol efficacy of B. subtulis over other microbial an­
AUDPC exhibited negative correlation to plant height, biomass and fruit tagonists in suppression of bacterial wilt under greenhouse (Yang
yield. There was a strong and significant negative correlation between et al., 2012; Almoneafy et al., 2014; Bhai et al., 2019) and field
crop mortality (r = −0.78, p = 0.014; r = −0.90, p = 0.001), disease conditions (Guo et al., 2004) have been reported. Guo et al. (2004)
incidence (r = −0.77, p = 0.016; r = −0.94, p<0.001), severity also observed significantly high biocontrol efficacy for Serratia spp.
(r = −0.32, p = 0.399; r = −0.44, p = 0.240), and AUDPC and fluorescent Pseudomonad. Nevertheless, a corresponding field
(r = −0.84, p = 0.005; r = −0.90, p = 0.001) to fruit yield in ex­ trial conducted by Xue et al. (2012) revealed considerably high
periment 1 and 2, respectively (Table 5 and 6). biocontrol efficacy of Serratia spp. both under greenhouse and field
Plant height and biomass also depicted a similar association to crop experiments. Similarly, moderate biocontrol potency had also been
mortality, disease incidence, severity and AUDPC. However, a strong po­ reported for Acinetobacter spp. in the management of bacterial wilt
sitive correlation was observed between plant growth and yield attribute (Xue et al., 2009).
components. Significant and highest positive correlation among the plant Biocontrol potency of Trichoderma spp. in in-planta suppression of
growth and yield attributes was displayed between plant height and fruit bacterial wilt has also been reported. According to Abd-El-Khair and
yield (r = 0.70, p = 0.035; r = 0.90, p = 0.001), even though biomass Seif El-Nasr (2012), the incorporation of T. hamatum and Bacillus spp.
also exhibited strong association with fruit yield (r = 0.55, p = 0.128; significantly reduced bacterial wilt incidence and severity under
r = 0.83, p = 0.005) in experiments 1 and 2, respectively (Table 5 and 6). greenhouse and field conditions. The authors observed that B. subtilis
consistently exhibited higher biocontrol potency, though T. hamatum
exhibited complete protection and superior biocontrol efficacy against
4. Discussion bacterial wilt in one of the four experiments. Elsewhere, crop mortality
to bacterial wilt of eggplant was significantly reduced in plots treated
4.1. Efficacy of microbial antagonists on bacterial wilt of tomato and subsequently planted with seedlings previously raised in nurseries
incorporated with T. harzianum (Nahar et al., 2019). Similarly, com­
Soil drenching with antagonistic bacteria and fungi effectively parable findings were observed by Kariuki et al. (2020) who reported
reduced bacterial wilt incidence, crop mortality and AUDPC in both

Table 5
Correlation coefficients between crop mortality, incidence, severity, area under disease progress curve (AUDPC), plant height and yield attributes in experiment 1.

Crop mortality Incidence Severity AUDPC Plant height Biomass

Incidence 0.96** –
Severity 0.23 0.28 –
AUDPC 0.93** 0.98** 0.27 –
Plant height -0.40 -0.26 -0.08 -0.35 –
Biomass -0.58 -0.71* -0.07 -0.74* -0.02 –
Fruit yield -0.78* -0.77* -0.32 -0.84** 0.70* 0.55
*
Significant at the 0.05 probability level
**
Significant at the 0.01 probability level

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A.O. Wamani, J.W. Muthomi, E. Mutitu et al. Journal of Natural Pesticide Research 6 (2023) 100051

Table 6
Correlation coefficients between crop mortality, incidence, severity, area under disease progress curve (AUDPC), plant height and yield attributes in experiment 2.

Crop mortality Incidence Severity AUDPC Plant height Biomass

**
Incidence 0.93 –
Severity 0.37 0.51 –
AUDPC 0.94** 0.98** 0.63 –
Plant height -0.82** -0.83** -0.17 -0.75* –
Biomass -0.81** -0.89** -0.67* -0.89** 0.57 –
Fruit yield -0.90** -0.94** -0.44 -0.90** 0.90** 0.83**
*
Significant at the 0.05 probability level
**
Significant at the 0.01 probability level

superior biocontrol potency of diverse species and isolates of Tricho­ accumulated more heat that deleteriously affected infection and es­
derma in suppressing bacterial wilt in field-grown tomatoes. tablishment of the fungal biocontrol agents. Pietikainen et al. (2005)
The biocontrol potency of an antagonistic isolate against bacterial reported that the activity of fungi substantially reduced in soils with
wilt significantly depends on its ability to secrete inhibitory substances temperatures above 30 °C and was nonexistent at 40 °C while bacteria
such as antibiotics, siderophores, cell wall degrading enzymes, com­ were still active in soils with temperatures as high as 45 °C.
petition for sites on the root surface and induction of systemic re­ A high population of soil bacteria can significantly reduce the effi­
sistance (Seleim et al., 2011). Biocontrol potency of B. subtilis in cacy of both fungal and bacterial biocontrol agents by reducing their
managing bacterial wilt has been previously associated with the pro­ establishment through competition for limited space and resources
duction of siderophores and surfactin which are instrumental in sup­ (Stewart et al., 2010; Mazzola and Freilich, 2017). In the current study,
pressing the causative pathogen, R. solanacearum (Yang et al., 2012; preliminary investigations revealed that the experimental site harbored
Chen et al., 2013). However, in planta experiments suggested that the high bacteria population, approximately 4.5 × 105 CFUs/g of soil. The
ability of B. subtilis to form robust root-associated biofilms primarily high biomass of resident soil microbial inhabitants was found to sig­
contributed to its superior biocontrol potency through the improvement nificantly contribute to the decline in biocontrol efficacy of T. har­
of cell colonization and increased antibiotic concentration (Chen et al., zianum Bae and Knudsen (2005).
2013).
Mycoparasitism, antibiosis, and competition for space and nutrients 4.2. Effects of microbial antagonists on growth and yield of tomato
are some of the biocontrol mechanisms reported for Trichoderma spp. in
the suppression of phytopathogens (Redda et al., 2018). Also of equal Microbial antagonists also exhibited varying levels of plant growth
importance is the induction of host plant resistance by the biocontrol promotion and yield increment compared to untreated control.
agents. Konappa et al. (2018) reported an increase in the activity of Trichoderma spp. and rhizobacteria with biocontrol potential have
defense-related enzymes after treating tomato plants with Trichoderma previously been found to increase plant growth and yield of diverse
spp. to manage bacterial wilt. Similarly, Zhou et al. (2021) also recently crops treated for the management of diverse soil-borne diseases (Pirttilä
observed an increase in the activity of defense enzymes, superoxide et al., 2021). Also, variation in plant growth promotion and yield in­
dismutase, peroxidase, and polyphenol oxidase when treated with a crement by biocontrol agents for the management of bacterial wilt
single dose or consortia of T. virens and B. subtilis. under field conditions have been reported (Almoneafy et al., 2014;
Overall, the biocontrol potency of bacterial antagonists exhibited Singh et al., 2016; Kariuki et al., 2020). Kariuki et al. (2020) recorded
less fluctuation compared to that of fungal antagonists. Abiotic and superior efficacy of Bacillus spp. and Trichoderma spp. in increasing
biotic factors can significantly influence the performance of microbial tomato yield when incorporated for management of bacterial wilt under
biocontrol agents (Stewart et al., 2010; Moosavi and Zare, 2015). The field conditions.
basic soil pH recorded in the experimental site could have favored the Trichoderma hamatum, T. viride, and B. subtilis exhibited comparably
survival and establishment of bacterial antagonists over fungal an­ higher and consistent results in biomass accumulation and yield in­
tagonists. Growth and proliferation of bacteria are highly favored in crement in both experiments. In concurrence with the findings herein,
soils with near-neutral pH whereas high soil pH is an impediment to diverse species of Trichoderma (Kariuki et al., 2020) and T. viride
fungal growth and proliferation (Rousk et al., 2009; Wang et al., 2017). (Sharma and Kumar, 2009) exhibited significantly higher yield output
Stewart et al. (2010) reported that the biocontrol potency of Tricho­ compared to untreated control. Similarly, the effectiveness of Bacillus
derma spp. was highly favored by low soil pH (< 7) and significantly spp. and diverse strains of B. subtilis in promoting plant height, biomass
constrained in alkaline conditions. In contrast, they observed increased and yield increment has been reported (Almoneafy et al., 2014; Singh
biocontrol potency of P. fluoresces against take-all of wheat with in­ et al., 2016). Xue et al., (2009, 2012) also observed that incorporation
creasing soil pH. of Acinetobacter spp. and Serratia spp. in the management of bacterial
Reduction in biocontrol potency of Trichoderma spp. exhibited in wilt of tomato and pepper under field conditions resulted in biomass
experiment 2 can be attributed to increased soil temperature. and yield increment compared to untreated control. Likewise, a lack of
Cumulatively, experiment 2 received approximately 23.5 mm less considerable differences in height promotion in crops treated with
rainfall and 3.2 °C higher maximum temperature, compared to experi­ different antagonistic isolates has also been observed (Nawangsih et al.,
ment 1. However, it is unlikely that differences in rainfall amount 2012).
significantly influenced biocontrol efficacy given that inadequacy in Plant growth and yield increment associated with microbial bio­
soil moisture was supplemented through regular irrigation between the control agents can be conferred either directly through secretion of
two experiments. Nonetheless, the maximum ambient temperatures of growth regulators and facilitation of nutrient uptake or indirectly
up to 27.0 and 30.9 °C and average ambient temperatures of 19.0 and through prevention and reduction of harmful effects of plant pathogens
22.4 °C were recorded in experiments 1 and 2 respectively. (Seleim et al., 2011; Ab Rahman et al., 2018). Further, antagonistic
Though soil temperatures were not recorded, a previous study in isolates with the highest potency in reduction of R. solanacearum in­
Kenya showed that soils can warm by up to 23.4 °C higher compared to oculum in the rhizosphere also exhibited the highest reduction in
ambient temperatures (Mace, 2012). Subsequently, there is a high bacterial wilt incidence and severity compared to untreated control
likelihood that the black cotton soils characteristic of the study site (Kariuki et al., 2020).

7
A.O. Wamani, J.W. Muthomi, E. Mutitu et al. Journal of Natural Pesticide Research 6 (2023) 100051

4.3. Relationship between bacterial wilt incidence, crop mortality, severity, Declaration of Competing Interest
plant height and yield attributes
The authors declare no competing interest.
The measured and computed disease parameters under the current
study exhibited a significant positive correlation among each other and Acknowledgment
a significant negative association to plant height and yield attributes of
tomato crops. Comparable results have been reported in related studies. The authors would like to thank Thomas Thome for providing his
Nsabiyera et al. (2012) observed a strong positive correlation between farm for experimentation and technologists at the Department of Plant
incidence and severity of foliar and wilt diseases of Capsicum annum and Science and Crop Protection, University of Nairobi and the Department
a strong negative correlation between the disease parameters to total of Agricultural Science and Technology, Kenyatta University for their
fruit yield. Similarly, Chakraborty et al. (2021) reported that plant technical assistance in laboratory work.
height and total grain yield of rice treated with Fluorescent Pseudo­
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