Journal Pre-proofs

Evaluation of oil dispersion formulation of nematophagus fungus, Pochonia

chlamydosporia against root-knot nematode, Meloidogyne incognita in cu‐
cumber

N. Swarnakumari, R. Sindhu, G. Thiribhuvanamala, V. Rajaswaminathan

PII: S1226-8615(20)30736-6
DOI: https://doi.org/10.1016/j.aspen.2020.10.008
Reference: ASPEN 1634

To appear in: Journal of Asia-Pacific Entomology

Received Date: 19 October 2019

Revised Date: 8 October 2020
Accepted Date: 12 October 2020

Please cite this article as: N. Swarnakumari, R. Sindhu, G. Thiribhuvanamala, V. Rajaswaminathan, Evaluation
of oil dispersion formulation of nematophagus fungus, Pochonia chlamydosporia against root-knot nematode,
Meloidogyne incognita in cucumber, Journal of Asia-Pacific Entomology (2020), doi: https://doi.org/10.1016/
j.aspen.2020.10.008

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© 2020 Korean Society of Applied Entomology. Published by Elsevier B.V. All rights reserved.
1 Evaluation of oil dispersion formulation of nematophagus fungus, Pochonia chlamydosporia
2 against root-knot nematode, Meloidogyne incognita in cucumber

3 N. Swarnakumari1, R. Sindhu1, G. Thiribhuvanamala2 and V. Rajaswaminathan2

5 1.Department of Nematology, Tamil Nadu Agricultural University, Coimbatore 641003, India

6 2.Department of Plant Pathology, Tamil Nadu Agricultural University, Coimbatore 641003, India

7 E-mail of corresponding author: [email protected]

8
9 Short title: Bio-efficacy of P. chlamydosporia against M. incognita in cucumber

10 Highlights

11 Native isolate of P. chlamydosporia had nematode egg parasitization ability.

12 The oil formulation of P. chlamydosporia prepared was stable up to 90 days.

13 The formulation reduced M. incognita infestation in cucumber.

14 Abstract:

15 Root-knot nematode, Meloidogyne incognita is considered as one of the major non-insect pests of
16 crops. The management of these root feeders becomes highly challenging due to a strong host-
17 parasitic relationship. Pochonia chlamydosporia is a nematophagus fungus that colonizes eggs of
18 nematodes. This study aimed to test the efficacy of P. chlamydosporia (NAIMCC-SF0039) against M.
19 incognita. An oil dispersion formulation of P. chlamydosporia was prepared using emulsifiers and
20 vegetable oil. This formulation had a shelf-life of 90 days (3.3x108 CFU / mL) at room temperature (28
21 ± 1 °C). The inhibitory effect of oil formulation was tested against M. incognita by inoculating it on the
22 egg mass. We found that colonization of the gelatinous matrix occurred on the third day of inoculation
23 followed by complete egg parasitization on the seventh day. A greenhouse trial was laid out to
24 evaluate the biocontrol potential of P. chlamydosporia in cucumber (Cucumis sativus). The results
25 showed that the application of talc formulation of P. chlamydosporia at the rate of 1 kg per acre during
26 planting, followed by delivery of 1L of oil dispersion formulation through drip lines at 30-day intervals
27 caused the highest reduction of nematode infestation. This treatment recorded 67.9 and 57.5 %
28 reduction in egg masses and soil nematode population respectively than that of control.

29

30 Key Words: Pochonia chlamydosporia, Meloidogyne incognita, oil dispersion formulation, efficacy,
31 cucumber.
32 Introduction

33 Root-knot nematode, Meloidogyne sp. is an important parasite of horticultural crops. It has a wide
34 host range that includes field crops, vegetables, fruit crops, and medicinal plants. It penetrates the
35 root and gets established in vascular region. The feeding site of Meloidogyne spp. is called as giant
36 cells, which form root-knot galls on feeder roots of plants. Among various vegetables, cucumber is the
37 most susceptible crop to root-knot nematode and damage may extent up to 30% (van der Wuff ,
38 2010). The yield loss caused by Meloidogyne spp. ranges from 6% to 18% in cucumber (Manjunatha
39 et al., 2017). Only a few carbomate and organophosporus nematicides are available which will also
40 be withdrawn soon from the market due to their hazardous effect on the soil ecosystem. Biological
41 control is one of the ecofriendly methods of nematode management. The nematophagus fungus,
42 Pochonia chlamydosporia (Zare and Gams, 2001) is a proven bio-control agent of root-knot nematode
43 (Manzanilla-Lopez et al., 2013). It produces chlamydopsores and also possess endophytic nature and
44 act as saprophyte in the absence of nematode hosts. This fungus infests the eggs and females of
45 economically important species of plant parasitic nematodes such as root knot nematode
46 Meloidogyne sp. (Hidalgo-Diaz, et al., 2000), cyst nematodes Globodera sp. and Heterodera sp.
47 (Kerry and Crump, 1977).The fungus colonizes the nematode eggs by penetrating the eggshell within
48 ten days of inoculation (Kumari and Kalaiarasan, 2017). Cucumber is one of the important crops
49 grown under protected condition in India. This crop succumbs to root-knot nematode attack due to the
50 microclimate of the greenhouse. Ceiro-Catasúe et al. (2018) studied the effect of P. chlamydosporia
51 on M. incognita in cucumber and reported that the gall index got reduced to less than 2 in plants
52 protected with P. chlamydosporia. They stated that this fungus is a suitable alternative for long-term
53 control of nematode due to its resting structure. The results of our previous study confirmed that the
54 fungus can colonize up to a depth of 6 cm in various soil types (Sindhu et al., 2019). Based on these
55 results, a greenhouse trial was carried out in a nematode-infested field to assess the efficacy of oil
56 dispersion formulation of P. chlamydosporia (NAIMCC-SF0039) in cucumber. The methodology
57 adopted in this study and the results obtained are elaborated in this paper.

58 Materials and methods:

59 Culture of Pochonia chlamydosporia

60 Fungal culture of P. chlamydosporia (NAIMCC-SF0039) was obtained from the Department of

61 Nematology, Tamil Nadu Agricultural University, Coimbatore, India and sub-cultured on Potato
62 Dextrose Agar (PDA) medium under aseptic condition. The molecular identity of the fungus was
63 confirmed by ITS (forward and reverse sequence) which showed 98.76 % identity with P.
64 chlamydosporia var. spinulospora (AB758254).

65 Preparation of oil dispersion formulation of P. chlamydosporia

66 Oil dispersion formulation of P. chlamydosporia was prepared using combinations of emulsifiers,

67 vegetable oil and surfactants (Table 1) as per the method described by Mbarga et al.( 2014). Conidial
68 suspension was prepared by filtering the macerated fungal mat using two layers of sterile muslin
69 cloth. The spore count was determined using haemocytometer (Neubauer). This conidial suspension
70 was then added to the above mentioned homogenized mixture.

71 Table 1. Composition of oil dispersion formulation of P. chlamydosporia

Ingredients Percentage (%)

Sunflower oil 74
Triton X-100 15
Tween 80 5
Glucose 4
Conidia 2
72
73 Testing shelf-life
74
75 Conidial viability of oil dispersion formulation was tested by plating the formulation at different intervals
76 viz., 15, 30, 60 and 90 days of preparation. About 1 mL of oil dispersion was transferred to a
77 microfuge tube and centrifuged for 5 min at 2000 rpm. The supernatant was discarded and oil
78 droplets were eliminated by using sterile filter paper. Then, the conidial spores were re-suspended in
79 sterile water and Tween-80 (0.05%). The mixture was vortexed for 2 minutes and 1 mL was
80 transferred to sterile Petri plate (99 mm). Then, 15 mL of sterile-molten cooled PDA was poured into
81 these plates. The plates were then incubated at 25 °C for the next five days.
82
83 Testing parasitization potential of P. chlamydosporia
84
85 Egg masses of M. incognita were collected from infested roots and surface sterilized using sterile
86 water for 5-10 seconds followed by 1% streptomycin sulphate. It was again washed thrice with sterile
87 water. After surface sterilization, single egg mass was placed at the center of an agar (1%) plate.
88 Subsequently, conidial suspension (500 μL) was inoculated on egg mass in Petri plates. These plates
89 were incubated at 25 °C for seven days. Observations were taken every alternate day.
90
91 Testing the effect of ingredients in the formulation on M. incognita

92 A pot culture experiment was conducted to test the effect of ingredients used for preparation of oil
93 dispersion formulation on M. incognita in cucumber. Sterilized soil was filled in pots (2.5 kg capacity)
94 and ingredients such as sunflower oil, Triton X-100, and Tween-80 were mixed in the top layer of soil
95 (15 cm) at the rate of 5 mL per pot. Cucumber seedlings were planted in the pots. The second stage
96 juveniles (J2) of M. incognita were inoculated into the pots ten days after planting. Nematode
97 population in soil and root was assessed 30 days after planting.
98
99 Bio-efficacy of P. chlamydosporia against M. incognita under greenhouse condition in cucumber
100
101 Root-knot nematode infested greenhouse located at Pogalur village (11.2571624 °N and 77.0381928
102 °E), Tamil Nadu, India was selected for experimental trial. This greenhouse contained black cotton
103 soil (silt 7.5%, clay 43.4%, sand 24.55%; pH 7.79; EC 0.28), which was rich in available potash (765
104 kg / ha). The initial nematode population (229 J2/ 250 g soil) was assessed by Cobb’s wet sieving and
105 modified Baermann’s funnel method (Southey, 1986). Treatments such as talc-based formulation (1 x
106 108 CFU / g) alone and also in combination with oil dispersion (7.5 x108 CFU / mL) formulation of P.
107 chlamydosporia were imposed to soil. There were four treatments with five replications. The
108 experimental plots were formed with a size of 22 x 4m2. Nematode population in soil and root were
109 assessed at the time of harvest. Gall-index (GI) was calculated based on the number of galls present
110 in single plant root system (Barker, 1985). The index was ranged between 1 to 5 (GI 1 -1-20 galls; 2 -
111 21-40 galls; 3 - 41-80 galls; 4- 81-90 galls; 5 - 90-100 galls / 5g root).
112
113 Statistical analysis
114 Statistical analysis was performed using ANOVA and DMRT, as described by Sakal and Rohlf (1995).
115
116 Results
117
118 Oil dispersion formulation of P. chlamydosporia
119
120 Oil dispersion formulation of P. chlamydosporia was prepared by combining vegetable oil and
121 emulsifiers. Parasitization, shelf life and bio efficacy of the formulation were tested. The oil dispersion
122 formulation was prepared using sunflower oil. The quantity was calculated based on the HLB value.
123 Observation showed that the growth of the fungus was completely stopped by the additives and spore
124 load was stable up to 90 days of storage with negligible reduction in colony-forming unit (CFU) count.
125 The formulation showed conidia and mycelial hyphae along with oil droplets when observed under
126 compound microscope. The spore load was 7.5 ×108 CFU / mL. The shelf-life of oil dispersion
127 formulation was recorded at 15, 30, 60 and 90 days of storage by recording the CFU. The spore load
128 was 7.5 ± 5×108 CFU / mL at the time of preparation and subsequently got reduced with passage of
129 time (Fig. 1).
131 Fig. 1. Viability of P. chlamydosporia conidial spores in oil dispersion formulation at different days of
132 storage

133
134
135 Oil dispersion formulation was inoculated on eggs and observed for colonization. The gelatinous
136 matrix of egg mass was completely colonized on the third day of inoculation and it turned black. The
137 hyphae started penetrating on the fourth day of inoculation by forming an appressorium on eggshell
138 (Fig. 2). The complete colonization of eggs was observed on the seventh day of inoculation.
139
Fig. 2. Hyphal penetration and colonization of P. chlamydosporia on root-knot nematode eggs

140
141 Effect of oil and other ingredients of formulation on M. incognita in cucumber
142
143 The ingredients used for preparation of oil-based formulation were tested for its efficacy against M.
144 incognita under pot culture condition. The results showed that the ingredients such as sunflower oil,
145 Triton X-100, Tween-80 and glucose did not have any influence on nematode infestation (Table 2).
146
147 Table 2. Effect of ingredients of oil dispersion formulation on M. incognita infestation in cucumber
Ingredients Nematode population Shoot height (cm)
(5 mL per 2.5 kg soil) Soil / 250 g Root-knot galls / plant
T1 – Sunflower oil (74%) 123.5 27.50 33.0
T2 – Triton X-100 (15%) 127.2 27.25 33.5
T3 – Tween-80 (5%) 125.3 26.25 33.8
T4 – Glucose (4%) 123.8 28.25 32.5
T5 – Control (Untreated) 126.5 28.75 32.3
CD (p=0.05) 3.26 3.25 3.26
NS NS NS
148 NS – Non significant
149
150 Bio-efficacy of P. chlamydosporia against M. incognita under greenhouse in cucumber
151
152 Effect of P. chlamydosporia on M. incognita was assessed under nematode infested greenhouse.
153 Observations revealed that the beds treated with talc-based formulation of P. chlamydosporia at the
154 rate of 1 kg / acre during planting followed by delivering 1L of oil dispersion formulation through drip
155 lines at 30-day interval caused the highest reduction in nematode infestation (Table 3 ; Fig. 3). The
156 number of egg masses per root system was reduced by 67.9 % than that of control. Similarly, soil
157 nematode population (J2) was reduced to the extent of 57.5 % than that of control. P. chlamydosporia
158 treatments had a significant influence on the yield too, showing a 69.1 % than that of control.
159
160 Table 3. Inhibitory effect of oil dispersion formulation of P. chlamydosporia on M. incognita infestation
161 in cucumber under greenhouse
162
Number of J2 / 250 g Number of egg Gall- Yield (kg / plot)
Treatments
soil masses / 5 g root index (22 x 2 m2)
% % %
reduction reduction increase
T1 - SA of P.c at 2.5 kg ac-1 at 274.8b 37.9 56.8b 33.5 4 490.4b 27.9
the time of planting. (2.44) (1.75)

T2 - SA of P.c at 1 kg at the 250.4a 43.4 46.8b 45.2 4 530.4b 50.0

time of planting +1.5 kg ac-1 at (2.39) (1.67)
30 DAP

T3 - SA of P.c at 1 kg at 199.2a 57.5 27.4a 67.9 3 598.0a 69.1

planting + oil dispersion (2.29) (1.44)
formulation on 30th, 60th and
90th DAP at 1L ac-1 for each
 interval

T4 - Untreated control 442.8c - 85.4c - 4 353.6c

(2.65) (1.93)

CD (p=0.05) 0.11 0.15 49.9

163 *SA of P.c - Soil application of talc-based formulation of P. chlamydosporia DAP - Days after planting
164 Figures in parentheses are log transformed values. Means followed by same alphabets are not significantly different from each
165 other as per DMRT.
166
Fig. 3. Inhibitory effect of P. chlamydosporia formulation on M. incognita infestation in cucumber

UT T

UT – Untreated Presence of small galls at 90 DAP in

T- P. chlamydosporia Treated treated plant roots

167 Discussion
168 The nematophagus fungus, P. chlamydosporia is a proven egg parasitic fungus. It penetrates the
169 eggshell and colonizes egg contents (Kumari and Kalaiarasan, 2017). The results obtained and the
170 probable reasons for lower nematode infestation are discussed below.
171
172 Oil dispersion formulation
173
174 Any nematicidal formulation delivered through drip lines provides easier solution for nematode
175 problems in a greenhouse. Nematodes are major parasites of horticultural crops and most of them are
176 grown under drip irrigation. The formulation prepared in current study was tested in the laboratory to
177 assess the parasitization ability and was found to be effective. This study is supported by the findings
178 of Prusty Abhishek and Mahalik (2018) who have reported that the application of liquid formulation of
179 P. chlamydosporia at the rate of 10 mL / kg of soil showed considerable reduction in root-knot
180 nematode infestation in brinjal. This study shows that the only major constraint in the oil-based
181 formulation is the immiscibility of oil with conidial suspension. It requires a thorough shaking of
182 formulation before each application. The ingredients used for preparation of oil dispersion was tested
183 on M. incognita infestation, and it did not show any inhibitory effect. In contrast, finding of (D’Addabbo,
184 Laquale et al., 2019) showed that application of sesame oil reduced soil nematode population and gall
185 index. In the current study the ingredients were released to the soil in meager quantities hence did not
186 show any nematicidal properties.

187 Bio-efficacy of oil dispersion formulation

188 Various formulations of P. chlamydosporia are used by vegetable growers for the management of
189 phytonematodes (Damme and Veerle Van, 2005; Inomoto et al., 2006). Findings of Sharf et al.
190 (2014) confirmed that the application of P. chlamydosporia mycelium showed higher reduction of M.
191 incognita infestation in common bean compared to application after the nematode inoculation. Similar
192 observations were recorded by Cláudia et al. (2011) who reported that pre-plant application of P.
193 chlamydosporia at the rate of 30 g / m2 recorded the lowest nematode population in the soil in terms
194 of eggs and juveniles. Jiue-In Yang et al. (2012) found that P. chlamydosporia colonization resulted in
195 a 50% reduction of egg masses, juveniles and galls. Kerry et al. (1993) stated that pre-plant
196 application of P. chlamydosporia resulted in control of long term nematode damage. This result is also
197 in accordance with Affokpon et al. (2015) who recorded that the pre-plant application of P.
198 chlamydosporia resulted in 50% reduction of eggs and 25% reduction in galling and multiplication.
199 Findings of Zakaria, et al. (2013) showed that the combined application of P. chlamydosporia by
200 drenching chlamydospores and culture filtrate Photorabdus luminescens as single or joint treatments
201 reduced nematode infestation in cucumber under microplot condition. Falling in line with the above
202 findings, the results of this study also revealed that the pre-plant application of talc-based formulation
203 followed by oil dispersion in split doses caused lowest infestation compared to other treatments. In
204 this study oil dispersion formulation which is unique and was tested under greenhouse using drip
205 lines.
206 This may be due to the following reasons;
207 (1) The initial soil egg population was colonized by pre-plant application.
208 (2) Application of oil dispersion formulation 30 days after planting (DAP) colonized the first
209 generation egg masses and subsequent generations by the application at 60 and 90 DAP.
210 (3) The endophytic nature of the P. chlamydosporia protected the roots and prevented entry
211 of freshly hatched juveniles and also promoted growth, which reflected in higher yield.
212 Egg masses produced at the time of harvest were reduced by 67.9% compared to that of control,
213 which indicated that P. chlamydosporia was unable to eradicate nematode population in one season.
214 The percentage of reduction could be increased if the same sets of treatments are imposed for two
215 more crops in the same field.
216
217 Conclusion
218
219 The study confirms that the native isolate of P. chlamydosporia possesses highest biocontrol
220 potential. The formulation prepared in this study had only one disadvantage which is the immiscibility
221 of oil and water phases and settling of conidial spores during storage. These problems need to be
222 addressed and resolved for commercial exploitation. There is a scope for research to test the potential
223 of P. chlamydosporia against other phytonematodes also.
224
225 Declarations
226 Ethics approval and consent to participate
227 Not applicable.
228 Consent for publication
229 Not applicable.
230 Competing interests
231 The authors declare that they have no competing interests.
232
233 Funding:
234 No separate funding was allocated for this project. Laboratory and infrastructure support was provided
235 by Tamil Nadu Agricultural University, Coimbatore, Tamil Nadu, India.
236
237 Acknowledgement
238 The authors express their gratitude to the administrators of TNAU for providing laboratory and
239 glasshouse facilities to execute this research work. Our special thanks to the farmer, Mr. Muthusamy
240 who provided the greenhouse to conduct the experiment.

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309 Highlights

310 Native isolate of P. chlamydosporia had an endophytic ability.

311 The oil formulation of P. chlamydosporia prepared was stable up to 90 days.

312 The formulation reduced M. incognita infestation in cucumber.

313

314 23.
315 Graphical abstract
P. chlamydosporia Oil dispersion Formulation mixing with water
formulation

P. chlamydosporia treated and untreated plants Delivery of formulation through

drippers

316

317 24.
318 Highlights

319 Native isolate of P. chlamydosporia had nematode egg parasitization ability.

320 The oil formulation of P. chlamydosporia prepared was stable up to 90 days.

321 The formulation reduced M. incognita infestation in cucumber.

322

323 25.