Bioremedy of Cotton Aphid (Aphis gossypii Glov.) (Hemiptera: Aphididae) by the Application of Different Fractions of Entomopathogenic Bacteria (Xenorhabdus Spp.)

Erum Yawar Iqbal1*, Ashfaque Ahmed Nahiyoon1, Shahnaz Dawar2 and Shahina Fayyaz1

1National Nematological Research Centre, University of Karachi, 75270- Karachi, Pakistan

2Dept. of Botany, University of Karachi, 75270- Karachi, Pakistan

ABSTRACT

Molecularly identified indigenous entomopathogenic bacteria (EPB) Xenorhabdus have been used to access their efficiency against cotton Aphid Aphis gossypii (Glov.). An efficient formulation of different parts of bacterial culture such as, cell-free supernatant, crude cell extract, bacterial culture and methanol extract in in vitro and green house condition have been exploited. The results have shown that different formulants of EPB produced significant effects on Aphid mortality, change in population density, population reduction and fecundity. Cell-free supernatant of new EPB species Xenorhabdus steinernematis n.sp. strain PAK. CB10 (KU097324) and the other X. indica PAK. S.B.56 (MF521953) resulted in both the highest mortality rate [(94.33±2.05, 100.00±0.00)%] @ 300µl/10ml at 30ºC and the lowest fecundity [(65.00±3.00) eggs/gravid female] in green house condition after 5 days of treatment. Crude cell extract of all bacterial fractions were found to be least effective as compared to cell-free supernatant, bacterial suspension and methanol extract suggesting that EPB has the potential to release its metabolites with insecticidal mode of actions in the surrounding culture media. New species proficiency was evaluated in comparison with other indigenous isolates and resulted in most efficient at 30ºC after 24h and proved to have good effectiveness as biological control agent and can be easily used instead of live bacteria in future formulations.

Article Information

Received 28 August 2019

Revised 07 September 2019

Accepted 27 September 2019

Available online 27 February 2020

Authors’ Contribution

EYI and AAN performed, analyzed and interpreted the aphids data regarding the biocontrol potential of bacteria. SF and SD supervised this research and helped in writing the manuscript.

Key words

Cotton aphid, EPB, Crude cell extract, Cell free Suspension, bacterial culture, methanol extract

DOI: https://dx.doi.org/10.17582/journal.pjz/20190828110853

* Corresponding author: erum_i@yahoo.com

0030-9923/2020/0003-0875 $ 9.00/0

Copyright 2020 Zoological Society of Pakistan

INTRODUCTION

Aphids possibly are important crop pests globally (Blackman and Eastop, 2000). Due to small size, high reproductive potential, cryptic nature of feeding and ability to act as vectors of plant viruses, they have dominating value (Clark and Perry, 2002; Davis et al., 2005). Aphids like other phloem feeding insects, can exhibit a considerable degree of host specificity. (Hooks and Fereres, 2006; Rasmussen et al., 2008; Catangui et al., 2009).

Biological agents or biocontrol agents, for controlling agricultural pests have been widely used for several years. This is partly due to the strict governmental regulations for the use of hazardous chemicals, as these chemicals can remain prolong in the food chain (Bro-Rasmussen, 1996; Fareeha et al., 2017). During last two decades, a number of entomopathogenic bacteria (ENB) have gained considerable interest as biological control agents. Gram-negative bacteria Xenorhabdus spp. that belongs to the family Enterobacteriaceae and symbiotically associated with entomopathogenic nematodes of the family Steinernematidae (Boemare et al., 1993; Forst et al., 1997) has proved composite relation between these bacteria and their nematodes. Their association has justified the severe toxicity against many insect species (Akhurst, 1983; Herbert and Goodrich-Blair, 2007; Herbert et al., 2009). Nematodes release the symbiotic bacteria from their intestinal tracts into the insect host’s heomocoel, as they infest their insect hosts, as a results of this action, death of insect hosts happened within 48 h, this is a resultant of the combined actions of bacterial propagation, septicemia, and toxins secretion (Bowen and Ensign, 1998; Sicard et al., 2004; Yang et al., 2012).

Both Xenorhabdus and Photorhabdus bacteria have been shown to grow successfully under laboratory conditions, and both cell suspensions and cell-free supernatants of these bacteria have been found to cause adverse effect and cause the death of several insect pests, such as desert locust Schistocerca gregaria (Forskål) (Mahar et al., 2004), red flour beetle Tribolium castaneum (Herbst) (Shrestha and Kim, 2010), wax moth Galleria mellonella (L.), beet armyworm Spodoptera exigua (Hübner), diamondback moth Plutella xylostella (L.), cotton leafworm moth Spodoptera littoralis (Boisduval) (Campos-Herrera et al., 2009) and black vine weevil Otiorhynchus sulcatus (Germar) (Mahar et al., 2008). Hence, these bacteria secrete their metabolic products that are toxic or immunosuppressive to the host insects cause the death of several important agricultural insect pests (Bowen et al., 2000; Chongchitmate et al., 2005; Grewal et al., 2005; Sharma et al., 2002; Mahar et al., 2004, 2008; Bode, 2009; Brivio et al., 2010). Effects of a protease inhibitor protein from Xenorhabdus bovienii on physiology of pea aphid (Acyrthosiphon pisum) was also determined (Jin et al., 2014).

Herein, this study was aimed to determine the aphicidal activity of whole and in fractions of indigenous and a new EPB species Xenorhabdus steinernematis n.sp. strain PAK. CB10 (Erum et al., 2019) formulants against Cotton Aphid.

 

MATERIALS AND METHODS

Cotton Aphids were obtained from the Pakistan Agriculture Research Council, Pakistan and were grown on a mixture of sawdust and soybean grain to establish a fresh spawn (Bussaman et al., 2009). A fresh aphid progeny in a glass bottle was used to maintain a pair of male and female at 28 °C for reproduction. This domestic bred was used for all experiments.

Entomopathogenic bacteria symbiotically associated with entomopathogenic nematodes were isolated from surface-sterilized infected Galleria mellonela larvae, (Table I). EPNs were obtained from culture stock of National Nematological Research Centre, University of Karachi maintained by Prof. Dr. Shahina Fayyaz and bacterial isolation method was followed, previously described by (Kaya and Stock, 1997).

Culturing of EPB

A nutrient bromothymol blue triphenyltetrazolium chloride agar (NBTA) medium, consisting of 37 g nutrient agar (Criterion, USA), 25 mg bromothymol blue powder (Lab-Chem, UK), 4 ml of 0.01 g/ml 2, 3, 5-triphenyltetrazolium chloride (Sigma-Aldrich, USA), in 1000 ml distilled water, was used to prepare culture plates for EPB (Lacey, 1997). The bacteria were spread onto NBTA plates, and the plates were sealed and incubated in the dark at 28 °C for 24h. These bacteria were found to form blue colonies on NBTA agar (indicated Phase I stage) (Stock et al., 1998; Grewal et al., 2005) and the colonies were selected and sub-cultured to acquire colonies with uniform characteristics. The selected colonies were individually grown in 25 ml of Luria Bertani (LB) broth (Sigma-Aldrich, USA) and placed in the incubator shaker (200 rpm/min) at 28 °C for 48h under complete darkness (Lacey, 1997). The concentration of whole bacterial cell suspension was determined by the plate-count technique (Klement et al., 1990) and adjusted to 1x104 colony-forming units per ml (CFU/ml) using sterile 1 g/L peptone solution. To obtain cell-free supernatant, cell suspension was centrifuged at 2500×g and 4 °C for 5 min and filtered using a 0.22-μm filter. The resulting bacterial pellets was collected and used for preparing crude cell extract dilutions. A total of 100 ml of LB broth was added to 1 g (approximately 1×104 cells) of bacterial pellets vortex for 1 min. these were then used as stock and for preparing further concentrations. Bacterial methanolic extract procedure was adapted from (Boszormenyi et al., 2009). For a single treatment 15.4 µg/ ml was examined for it efficeincy.

Treatment preparation of EPB

In order to determine the insecticidal potential of different EPB spp. strains, the bacterial culture (@ 104 CFU/ml), bacterial cell free supernatant and crude cell extract (100,200,300µl/10ml) and methanol extract were tested in in vitro and in vivo against aphids.

Imidacloprid (50 μg/mL) (Product code: 00231, Brand: SKY SEEDS) was used as positive control. As negative controls, sterile cell-free LB was used for comparison with the whole culture, supernatant and bacterial residual treatments.

Aphicidal activity of different Xenorhabdus species on mortality of cotton aphids

Effect of different EPB were investigated using one hundred adult aphid female (1 d old) transferred to each (10-mm diameter × 10 inches in height) glass jars for each treatment. A total of 10ml (104 CFU/ml) of bacterial culture, cell-free supernatant and bacterial crude cell extract and methanol extract (100, 200, 300 µl/10ml) were then sprayed onto the aphids separately. The same volumes of LB broth and Imidacloprid (a commercial aphicide) at the concentration of 0.04% were used as negative and positive control groups, respectively (four replications/treatment). All jars were covered with lids and placed in a temperature controlled chamber at 25, 30 and 35°C with 80% relative humidity in complete darkness. Aphids mortality was observed every 24 h for four consecutive days after treatment. The experiment was repeated twice.

 

Table I. Mortality rates of cotton aphids after treated with different fractions of EPB (crude cell extract, cell-free supernatant, bacterial culture and methanol extract) at different temperatures and durations.

Data are expressed as mean ± standard deviation (SD). Means within the same rows followed by the same lower case letters are not significantly different (P<0.05) as compared by LSD test; Means within the same columns followed by the same upper case letters are not significantly different (P<0.05) as compared by LSD test. CFS, Cell free supernatant; CCE, Crude cell extract; BC, Bacterial culture; ME, Methanol extract.

Mortality rate of Cotton Aphids was calculated using Abbot’s formula (Abbott, 1925), which compares living aphids in each treatment (At) with living aphids in each control (Ac):

Mortality (%)=(1−AtAc)×100

Greenhouse experiment

Single seed of cotton var. (TH-120/05 (Ghotki-1)) was sown in 10 ×10 inches earthen pots. After one month, pots were transferred in greenhouse net cage, when plants became infected with aphid as they reached the economic threshold level (ETL) i.e., 5-7 aphids per leaf (Afshari et al., 2009). All treatments were applied same as for in vitro experiment @ 50ml /plant by using handheld sprayer. Observation on three leaves /plant were randomly made from the top, middle and bottom (Kaushik et al., 1994).

Aphicidal activity of different Xenorhabdus species on fecundity of cotton aphids

Al living pregnant females were excised using sterile needle and number of eggs/gravid female recorded under stereomicroscope.

Statistical analysis

Mortality percentage of aphids in in vitro experiment was subjected to one way analysis of variance (ANOVA), SAS Institute, Cary, NC, USA] Significant differences between means of the treatment were determined using the least significant difference (LSD) test at P≤0.05. Green house experiment was designed in RCBD with 3 replicates for all treatments. Data were recorded after 1, 24 and 48h of treatment. Change in population density (CPD%) and Population Reduction percentage (PR%) in relation to untreated were calculated by (Henderson Tilton’s formula, 1955).

CPD % = (Xi-X)/X ×100

X=mean number of alive pest before treatment

Xi= mean number of alive pest after treatment

Where: Ta is Number of total pests after treatment, Tb is Number of total pests before treatment, Ca is number of pests in control after the time of treatment, Cb is number of pests in control before the time of treatment.

The data were subjected to ANOVA and LSD by using SPSS software.

 

RESULTS

Effects mortality of cotton aphids

Different formulants of EPB culture were found to induce mortality of cotton aphids at different levels (Table I). For all the bacterial treatments, the percentages of aphids mortality reached a maximum after 48h post-treatment and remained non- significantly unchanged afterward. Among all EPB, cell free supernatant of Xenorhabdus indica S.B.56 and Xenorhabdus steinernematis. C.B. 10 @ 300µl/10ml have shown best results (100.00±0.00) after 24h at 30ºC. X. indica S.B. 50 was found to be relative less efficient in performance and caused mortality of aphids up to (84.66±3.68) % after 24h @ 300µl/10ml, which was significantly different from other concentrations of same treatment as well as other treatments. Maximum morality caused by bacterial culture was (95.00±2.44, 94.00±1.41) % of Xenorhabdus indica S.B.56 and Xenorhabdus steinernematis C.B. 10, respectively after 24h at 30 ºC which is non-significantly different form the results of methanol extract of same species after 24h at 30 ºC (100.00±0.00, 94.00±1.41), respectively. Crude cell extract was found to be least effective and has shown significantly lower results than cell free suspension, bacterial culture and methanol extract of all studied EPB species as well as positive control (Imidacloprid). No dead aphids were observed after application with cell free LB broth.

Effects on different Xenorhabdus species on change in population density and population reduction

After 3 days of treatment, cell free suspension, bacterial culture and methanol extract of all EPB showed a maximum change in population density % (CPD %) along with population reduction % (PR %) of A. gossypii as compared to crude cell extract and negative control. Among all EPB species cell free suspension of Xenorhabdus steinernematis C.B. 10 @ 300µl/10ml have given significantly high results for CPD% and PR% (93.27%, 97.23%), respectively, followed by Xenorhabdus indica S.B.56 (78.76%, 74.56%). Bacterial suspension and methanol extract of Xenorhabdus steinernematis C.B. 10 and Xenorhabdus indica S.B.56 were rated second in their efficacy for CPD% i.e. (87.59%, 70.21%) and PR% (95.23%, 87.59%) of bacterial culture, CPD% (85.67%, 84.65%) and PR% (96.48%, 76.56%) for methanol extract. X. stokiae Pak. S.B. 65 was found to be least effective and have shown significantly reduced CPD% and PR% for all the fractions after 3 days of treatment. An overall performance of all the bacterial fractions (Fig. 1) represents that the Xenorhabdus steinernematis C.B. 10 @ 300µl/10ml was the highly effective one against the aphid, bacterial suspension and methanol extract.

 

Effects on fecundity of aphids

After 5 days of treatment, the reproduction of Cotton Aphids was found to decrease significantly after the exposure of all fractions of different EPB (Table II). Cell-free supernatant of Xenorhabdus indica S.B.56 X. indica S.B.50 and Xenorhabdus steinernematis C.B. 10 have shown significantly reduced aphids fecundity, at 25 C and 30 C as compared to 35 C. (Table III). Bacterial culture and methanol extracts of isolates S.B. 56 and C.B.10 have shown significant and equal to CCE results at 25 C and 30 C. Later on, CCE reduced the number of eggs/female to a significantly level lower than bacterial culture after 2 days. In contrast, crude cell extract of all EPB species showed least effect on aphids fecundity at all temperatures. Also, no aphids were found to survive after Imidacloprid application; hence, no fecundity was recorded. Even in negative control, (LB broth) fecundity of aphid was seems to be under the influence of temperature in which 25 C and 30C favors the relative high fecundity results.

 

Table II. Fecundity of cotton aphids after treated with different fractions of EPB (crude cell extract, cell-free supernatant, bacterial culture and methanol extract) at different temperatures in in vitro condition.

Bacterial strains	Bacterial fraction and Conc.	Fecundity (eggs/female)
		25°C	30°C	35°C
Pak.S.B.50	CFS
	100µl/10ml	65.00± 5.00aA	70.65± 6.59aA	100.66± 8.61aB
	200µl/10ml	65.00± 7.34aA	75.33± 5.64aA	90.33± 7.45aB
	300µl/10ml	70.33± 6.25aA	75.66± 6.59aA	89.66± 6.58aB
	CCE
	100µl/10ml	200.00± 10.28aC	200.33± 14.59aC	220.33± 20.51aC
	200µl/10ml	185.33± 12.36aC	225.66± 18.69bC	225.66± 13.45aC
	300µl/10ml	105.00± 12.86aB	130.22± 16.47aB	230.33± 11.36bC
	BC (104 CFU/ml)	115.00± 14.95aB	140.66± 13.72cB	100.66± 8.64aB
	ME	150.66± 17.26aB	100.66± 11.26cB	95.221± 6.48aB
Pak.S.B.56	CFS
	100µl/10ml	70.66± 7.58AA	75.33± 7.56aA	85.33± 7.69aB
	200µl/10ml	68.33± 4.67aA	82.66± 6.54aA	90.66± 6.59aB
	300µl/10ml	65.33± 5.69aA	89.33± 7.54aA	85.66± 7.68aB
	CCE
	100µl/10ml	155.33± 10.47aB	158.33± 14.12aB	120.33± 7.63aB
	200µl/10ml	150.66± 17.56aB	194.33± 11.45aC	150.68± 13.64aB
	300µl/10ml	149.66± 13.26aB	146.33± 14.16aB	155.33± 13.45aB
	BC (104 CFU/ml)	85.66± 9.48aA	100.66± 9.45aB	92.33± 7.15aA
	M.E	100.00± 14.57aB	115.33± 8.43aB	90.66± 6.14aA
Pak.S.B. 65	CFS
	100µl/10ml	190.33± 18.69aC	200.66± 15.46aC	160.33± 9.48aB
	200µl/10ml	200.66± 22.69aC	220.33± 14.26aC	220.66± 18.15aC
	300µl/10ml	200.33± 17.61aC	250.66± 18.45bC	268.33± 16.59bC
	CCE
	100µl/10ml	210.33± 15.24aC	250.33± 17.16bC	235.66± 12.58bC
	200µl/10ml	225.33± 24.26aC	260.33± 10.26bC	249.33± 17.26bC
	300µl/10ml	200.33± 12.69aC	265.66± 14.26bC	275.26± 15.34bC
	BC (104 CFU/ml)	185.66± 13.64aC	245.33± 11.26bC	200.33± 14.23bC
	M.E	175.33± 15.45aC	260.66± 14.23bC	210.33± 14.23bC
Pak.C.B.10	CFS
	100µl/10ml	80.33± 7.45aA	95.66± 6.45aA	120.33± 11.14aB
	200µl/10ml	95.66± 7.56aA	99.33± 9.15aA	122.66± 10.25bB
	300µl/10ml	60.26± 4.65aA	80.33± 5.64aA	125.33± 8.25bB
	CCE
	100µl/10ml	160.23± 6.65aA	135.33± 17.56bB	165.33± 7.62bB
	200µl/10ml	154.33± 7.56aA	145.33± 12.41bB	153.66± 6.54bB
	300µl/10ml	150.66± 6.12aA	159.33± 16.25bB	144.66± 10.26bB
	BC (104 CFU/ml)	80.66± 7.65aA	70.66± 4.36bA	110.66± 15.26bB
	M.E	75.66± 6.54aA	79.33± 7.16aA	125.66± 10.32bB
Imidacloprid	50µg/ml	0.00± 0.00cD	0.00± 0.00cD	0.00± 0.00cD
Luria Bertani Broth		336.33± 18.76dF	355.33± 20.56dF	300.66± 22.36dE

 

Data are expressed as mean±SD. Means within the same column followed by the same letters are not significantly different (P<0.05) as compared by LSD test. CFS, Cell free supernatant; CCE, Crude cell extract; BC, Bacterial culture; ME,Methanol extract.

 

DISCUSSION

Different formulants of EPB culture, particularly crude cell extract and methanol extract, demonstrated harmful effects on Cotton Aphids survival and fecundity.

 

Table III. Details of symbiotic bacteria used for controlling cotton aphids.

Strains	Bacterial symbionts	EPN	Accession No.	Authority*
Pak.S.B.50	Xenorhabdus indica	S. abbasi	MF498486	Shahina F. and Salma J.
Pak.S.B.56	Xenorhabdus indica	S. pakistanense	MF521953	Shahina F. and Salma J.
Pak.S.B. 65	Xenorhabdus stockiae	S. siamkayai	MF521964	Shahina F. and Salma J.
Pak.C.B. 10	Xenorhabdus steinernematis	S. maqbooli	KU097324	Shahina F. and Salma J.

 

* Accession No. authorized by these persons.

 

The high mortality rate caused by crude cell extract, bacterial culture and mewthanol extract of EPB suggest that toxic metabolites can transmit horizontally (most likely by direct contact) that is between infected aphids that come in contact with uninfected ones. Moreover, the decrease of fecundity of aphids caused by EPB proves that infection indicates vertical transmission of toxins effect between reproductive females and their offspring.

There are some reports of other microorganisms that have produced similar effects like aphids, such as Microsporidium phytoseiuli against Phytoseiulus persimilis Athias-Henriot and Wolbachia bacteria against Tetranychus urticae (Koch) (Bjørnson and Keddie, 2001; Vala et al., 2004). Cell-free supernatant of EPB in this study have shown to induce higher aphid M% and lower CPD%, PR% and fecundity than the other factions of bacteria. This may suggest that metabolites with insecticidal properties that were produced by EPB are more likely to be secreted to culture supernatant. Mahar et al. (2005) also found that X. nematophila cell-free metabolites required 4 days to kill 95% G. mellonella larvae whereas cell suspension needed up to 6 daya to induce 93% mortality. There are several reports indicated that Xenorhabdus sp. could produce and secrete several secondary metabolites with effective bioactivities such as benzylideneacetone (antibacterial compound), iodinine, phenethylamides, indole derivatives, xenorhabdins, xenorxides, and xenocoumacins (antibiotics), and primary metabolites, such as alkaline protease (Morgan et al., 2001; Caldas et al., 2002; Ji et al., 2004; Mohamed, 2007; Bode, 2009), whereby all of which are optional to play roles as insecticidal and immunosuppressive compounds. On the contrary, crude cell extract had least effect on aphid mortality and fecundity. This is probably due to the loss of important metabolites of bacterial crude cell extract during the withdrawal process, or they have never been there. Secondly, difference in the results of different EPB attributed the fact that different species of EPB have different protein density. It has previously determined that protein encoding density of different species of Xenorhabdus varied considerably (I1 Hwankim et al., 2017) The results clearly demonstrates that some toxic proteins are present in the crude cell extract which persist there after centrifugation and responsible for mortality but it was found to be least effective than other fractions of EPB. However, temperature dependency is also a valid factor affecting biological processes. The mean generation times, of A. gossypii, ranged from 17.43 (15°C) to 5.63 days (30°C) (Zamani et al., 2006). Below 25°C, this time significantly increased (Takalloozadeh, 2010).

 

CONCLUSIONS

In conclusion, it has been depicted that different parts of X. indica isolate SB50 and Xenorhabdus steinernematis C.B.10 culture produced varying effects on Cotton Aphids mortality and fecundity. As X. indica and Xenorhabdus steinernematis C.B.10 cell-free supernatant was found to be the most effective than its surfactant, this may advocate that X. indica isolate SB.50 and Xenorhabdus steinernematis C.B. 10 are more likely to secrete their metabolites with aphicidal activities to the adjacent culture media. This is measured to be imperative for future formulation of X. indica isolate SB.50 and Xenorhabdus steinernematis C.B. 10 as an environmental friendly biological control agent. More work is under process to formulate the supernatant of these bacteria and their effects under field conditions.

 

Acknowledgements

Our gratitude goes to Pakistan Science Foundation (PSF) for providing fund to conduct the Project (084) under which this research has been conducted.

 

Statement of conflict of interests

The authors declare that they have no competing interests.

 

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