Research Article

Insecticidal Potential of some Fermented Native Plant Extracts for the Management of White Grub (Scarabaeidae: Coleoptera) Infestation on Potato Tubers in Skardu Baltistan

Muhammad Ayub1*, Munir Hussain2, Sher Ahmad2, Syed Arif Hussain Rizvi3, Shahid Hussain1, Muhammad Qasim2, Muhammad Din2, Muhammad Ishaque Mastoi4 and Tajudin2

1PARC, Agricultural Research Station, Skardu, Gilgit Baltistan, Pakistan; 2Mountain Agricultural Research Centre, Juglote, Gilgit, Pakistan; 3Institute of Plant and Environmental Protection, National Agriculture Research Centre, Islamabad, Pakistan; 4Plant Sciences Division, Pakistan Agricultural Research Council, Islamabad, Pakistan.

Received | May 26, 2020; Accepted | January 12, 2021; Published | March 23, 2021

*Correspondence | Muhammad Ayub, PARC, Agricultural Research Station, Skardu, Gilgit Baltistan, Pakistan; Email: ayubskd2011@gmail.com

Citation | Ayub, M., M. Hussain, S. Ahmad, S.A.H. Rizvi, S. Hussain, M. Qasim, M. Din, M.I. Mastoi and Tajudin. 2021. Insecticidal potential of some fermented native plant extracts for the management of white grub (Scarabaeidae: Coleoptera) infestation on potato tubers in Skardu Baltistan. Pakistan Journal of Agricultural Research, 34(2): 259-265.

DOI | http://dx.doi.org/10.17582/journal.pjar/2021/34.2.259.265

Keywords | Plant extract, Insecticidal potential, Native plants, White grub, % infestation, Yield loss

Introduction

Potato (Solanum tuberosum L.) is the fourth major food crop of the world. In Pakistan, currently potato is grown on 109.7 thousand hectares producing 1938.1 thousand tonnes annually with an average yield of 17.7 tons/ha. (Agricultural Statistics of Pakistan, 2003-2004).

Potato is now gaining status of cash crop after Apricot in Gilgit Baltistan (Food and Agriculture Division, 2012). Its annual production is estimated 76,125.00 tonnes from an area of 3,045 hectares (Hashmi and Shafiullah, 2003) . Potato crop is attacked by a number of insect pests including wireworms, white grub, aphids, cutworm and others adversely affecting the yield of crop (Shakur et al., 2007). Among these insect pests, white grub is the most destructive pest damaging potato tubers and causes significant economic loss to potato growers of Gilgit-Baltistan.

White grubs are larvae of a Scarabid beetle in the order Coleoptera. There are about 1300 North American species and the world fauna of white grub exceeds 30,000 species (Mittal and Pajni, 2000). Several species of white grubs or scarabs are root feeding pests of grasses, grains, sugarcane, strawberry, potato tubers and young nursery plants (Crocker et al., 1996).

True white grubs are the larvae of May beetles (also called June Beetles) found in the genus Phyllophaga, of which there are over 100 different species. Phyllophaga larvae and other larvae of the family Scarabaeidae are often referred to as “white grubs”, including larvae of the Japanese beetle (Popillia japonica Newman), annual white grubs (Cyclocephala spp.) and the green June beetle (Cotinis nitida Lin.) (Selmen, 1998).

White grubs are root feeders and their adult beetles feed on the leaves of host plants (Musthak et al., 2013). White grubs are the most destructive and troublesome soil insects, impeding potato production especially in hilly states of India. The infected tubers have scooped out holes or half-eaten tubers which are rendered unfit for marketing (Chandel et al., 2015).

Reliance on injudicious use of synthetic pesticides to control pests has also given rise to a number of problems such as destruction of non-target beneficial organisms (parasitoids and predators) affecting food chain and biological diversity, secondary outbreaks of pests that are normally under natural control resulting in their rapid proliferation (Ouedraogo et al., 2011 ). There have also been cases of pests becoming tolerant to insecticides resulting in the use of double and triple rate of application (Stoll, 2000).

Although synthetic insecticides are used as the main controlling agent, optimal control cannot be obtained probably due to the development of insecticide resistance. Also, the uses of synthetic insecticides pollute the environment and can seriously affect the health of the farming communities . Therefore, search for natural product-based insecticides (botanicals), which are biodegradable and economically has become an area of extensive research. Resistance to botanicals does not develop as rapidly as to synthetic insecticides due to the structural complexity of natural toxicants (Silva et al., 2008). About 250,000 plant species in the world have been reported to possess compounds with insecticidal properties (Rafael, 2001).

The chemical insecticides are not effective in the white grub management system and these chemical pesticides are hazardous for human health and other organisms. Green plants are reservoir of effective and valuable natural source of pesticide (Hostettmann et al., 1997).

Interest in the use of bio-pesticides with selectivity towards phytophagous insects has increased in recent years, particularly in cropping systems that rely on natural enemies as a major component of integrated pest management (Rausell et al., 2000). In Gilgit-Baltisan (G-B) farmers are using toxic chemical insecticides injudiciously to control white grub.

Floral diversity in Gilgit-Baltistan is rich with wide spectrum of medicinal values but no effort has been made in this region to isolate, identify, and develop botanical pesticides. Rich biodiversity in G-B has potential opportunities to develop botanical pesticides formulation unit as in most parts of the Gilgit-Baltistan plant materials are also used traditionally to control insect pests (Ismail et al., 2014). Sophora alopecuroides found in Skardu (Baltistan) abundantly has the potential to commercialize as a natural pesticide against various insect pests (Rizvi et al., 2019).

In the area where this study has been conducted, farmers rely on toxic chemical insecticides injudiciously to control white grub. The massive use of synthetic insecticides in pest management program exerts hazardous effects on human health and environment. There is a dire need to adopt eco-friendly approach for pest management, therefore in this study fermented extracts of four readily available local plants were evaluated for their insecticidal potential against white grub infestation on potato crop.

Materials and Methods

Preparation of fermented plant extract

The aerial parts of native plants Sophora alopecuroides (called Khampa in local language “Balti”), Artemisia sieversiana (Khahuro), Peganum harmala (Isman) and Tagetes minuta (Suna Mindoq) were harvested at pre-blooming stage. The plant materials were washed thoroughly and then dried in shade. The dried plant materials were grinded to form fine powder and stored in hermetically sealed jars. Fermented extracts of four plants were prepared by dipping their powder in stream water in 25 kg capacity drums at 1: 9 w/v. Openings of drums were wrapped with muslin cloths and then placed in semi-shade for fermentation. Plant materials in drums were stirred daily. After two weeks fermented extracts were filtered through muslin cloth and then filter paper (Whatman No.1). These extracts were stored in glass jars placed in a cool dark place until field application.

Field application

Field experiments to evaluate the insecticidal effects of fermented extracts derived from four native plants against white grub were carried out at PARC, Agricultural Research Station, Skardu during two successive seasons of 2018 and 2019 under Randomized Complete Block Design with 13 treatments and 3 replications. Potato variety “Flamenco” was planted maintaining a plot size of 6.67m2 for each treatment with two 45 cm wide ridges of 5 meters length. Serial concentrations of fermented extracts were prepared by diluting @ 1, 3 and 5ml/ liter of water and drenching of this diluted extract was carried out @ 1000 l/ha on top of potato ridges in the evening hours whereas control plots were left untreated. Refree granules (Active Ingredient-Fipronil 0.3% G.) were applied during 2019 as standard control. Drenching was carried out twice in a season at one-month interval. First application of extracts was given on tuber formation initiated after one month of planting in mid-June. Total number of tubers and damaged tubers were recorded to determine % infestation. Number of white grubs found in each plot and weight of tubers per plot were recorded at harvesting time for assessment of yield and % yield loss.

Statistical analysis

All the statistical tests were conducted by using SPSS 17.0; P-values of less than 0.05 were considered significant. For the percent infestation, damage and yield data under field conditions were subjected to repeated measured analyses were subjected to simple analyses of variance. PROC GLIMMIX (SAS Institute, 2010) was used. All statistical analyses were conducted at the 0.05 level of significance.

Results and Discussion

Results obtained during 2018

Statistical analysis indicated highly significant difference in % tuber infestation between plots treated with S. alopecuroides fermented extracts at doses 3 and 5 ml/l and other plots. In plots treated with 1, 3 and 5 ml/l doses of T. minuta, A. sieversiana 1, 3 ml/l P. harmala and 1 ml/L S. alopecuroides fermented extracts moderate tuber infestations of 10.84 to 21.60 % were recorded. Results of this study revealed that drenching of S. alopecuroides fermented extract twice significantly reduced the white grub infestation on potato tubers to 1.67% and 4.05 % at concentrations of 5 and 3ml/l compared to untreated control plots and P. harmala extract 5 ml/l dose treated plots with significantly highest infestation of 24.06% (Table 1, Figure 1).

In case of number of white grub counted per plot at time of harvesting, there was significant difference among treatments (P=0.005) but there was no difference between treatments of S. alopecuroides extract doses of 3 and 5 ml/l @ 1000 l/ha. As shown in Table 1 from plots treated with S. alopecuroides extract 3 mL/L and 5mL/L lowest number of white grub 0.33 and 0.00 were captured, which significantly differed from other treatments during 2018. Without statistical difference, higher number of white grubs 7.67, 7.00 and 5.67 were recorded from control and plots treated with P. harmala extract at doses of 1 and 3 ml/l.

Similarly, there was significant difference in tuber yield obtained from plots treated with four fermented plant extracts and untreated control plots. Two applications of S. alopecuroides fermented extract at 5 ml/l significantly increased the tuber yield to maximum 27.64 t/ha followed by 19.74 t/ha tuber yield recorded from plots treated with 3 ml/l of same extract. Among other treatments including control, no significant difference was observed in tuber yield which ranged from 11.74 t/ha to 15.49 t/ha.

Results of this study revealed that infestation of white grubs on potato tuber caused significant loss of potato yield i.e., up to 58.40% but drenching of S. alopecuroides fermented extracts at 3 and 5 ml/l significantly reduced tuber yield loss to 28.58% and 5.31%.

Results obtained during 2019

Minimum infestation (3.47%) was recorded in standard control plots treated with Refree (Active Ingredient Fipronil 0.3% G) which did not differ significantly from 5.75, 3.85% infestation observed in plots treated with 3 and 5 ml/l of S. alopecuroides fermented extracts as shown in Table 2. Results indicate that the effect of synthetic pesticide used in the study has no significant difference with fermented extract of S. alopecuroides and A. sieversiana on reduction in white grub infestation. Statistically, %infestation in plots treated with fermented extracts of T. minuta, A. sieversiania and P. harmala did not vary significantly with each other but significantly differed from plots treated with S. alopecuroides fermented extracts (3 and 5 ml/l), A. sieversiana (5 ml/l), Refree (45 kg/ha) and control plots with significant difference receiving maximum infestation of 34.82%.

With no significant difference, lowest number of white grub 0.00, 0.33 and 0.67 were recorded in plots treated with 1 and 5 ml/l S. alopecuroides extract and Refree. Highest number of white grub 7.67, 8.67 and 8.33 were found in plots treated with extract dose of T. minuta 3ml/l, and P. harmala extract doses 1 and 3 ml/l respectively with no statistical difference. Higher number of white grubs i.e., 5.00 were recorded in untreated control plots which did not significantly differ from plots treated with fermented extracts of T. minuta, A. sieversiana, P. harmala at 1, 3 and 5 ml/l and S. alopecuroides 3 ml/l but significantly differed from plots treated with Refree 45 kg/ha and S. alopecuroides extract at 1 ml/l and 5 ml/l concentrations.

 

Table 1: Effect of Fermented extracts of four native plants on white grub infestation on potato under field condition during 2018.

Fermented extract	Dose mL/L	% infestation	No. of white rub/6.67m2	Yield t/ha	% yield loss
S. alopecuroides	1 3 5	10.84cde 4.047de 1.667e	3.00ab 0.33b 0.00b	15.49b 19.74ab 27.64a	43.96a 28.58ab 5.31b
T. minuta	1 3 5	20.07abc 12.35bcd 20.22abc	3.67ab 4.00ab 4.33ab	11.74b 14.24b 13.62b	57.51a 48.48a 51.93a
A. Sieversiana	1 3 5	20.75abc 16.70abc 14.97abc	4.00ab 3.67ab 3.00ab	12.75b 14.50b 14.74b	53.88a 58.40a 46.67a
P. harmalla	1 3 5	21.60ab 18.24abc 24.06a	7.00a 5.67a 3.67ab	12.39b 14.49b 13.99b	55.17a 47.57a 49.37a
Control	-	24.95a	7.67a	13.24b	52.88a

 

Data are the mean of three replicates and are represented as mean ± standard deviation. Means in the same column followed by the same lower-case letter are not significantly different at P = 0.05.

 

Table 2: Effects of Fermented extracts of four native plants on white grub infestation on potato tubers under field condition, 2019.

Fermented extract	Dose mL/L	% infestation	No. of white grub captured/plot	Yield t/ha	% yield loss
S. alopecuroides	1 3 5	15.00cde 5.75e 3.85e	0.33e 1.00de 0.67e	12.49c 18.49abc 23.99ab	50.51a 26.74abc 8.25bc
T. minuta	1 3 5	25.58abc 24.62abcd 27.58abc	5.33abc 7.67a 3.33bcde	13.99c 12.99c 11.10c	44.56a 48.52a 54.45a
A. Sieversiana	1 3 5	26.38abc 17.49bcde 10.52de	3.00bcde 2.33bcde 1.67cde	11.64c 14.49bc 16.24abc	53.90a 42.58ab 35.64abc
P. harmalla	1 3 5	31.35ab 32.09ab 29.64ab	8.67a 8.33a 6.33ab	12.99c 12.24c 12.10c	48.52a 51.49a 48.51a
Refree	45 kg/ha	3.47e	0.00e	25.24a	4.62c
Control	-	34.82a	5.00abcd	11.10c	56.03a

 

Data are the mean of three replicates and are represented as mean ± standard deviation. Means in the same column followed by the same lower-case letter are not significantly different at P = 0.05.

In case of potato tuber yield, maximum tuber yield of 25.24 t/ha was obtained from plots treated with Refree which did not differ significantly from 18.49 t/ha, 23.99 t/ha and 16.24 t/ha tuber yield, respectively recorded from plots treated with S. alopecuroides extracts at 3 and 5 ml/l and A. sieversiana extract at 5 ml/l doses. Minimum tuber yield of 11.10 t/ha obtained from the control and T. minuta 5 ml/l treated plots was not significantly different from tuber yields of 11.64 and 18.49 t/ha obtained from plots treated with S. alopecuroides extract doses 1 and 3 ml/l and T. minuta, A. sieversiana, P. harmala extracts at 1 ml/l, 3 m/l and 5 ml/l dose levels. Potato tuber yield of 16.24 ha/t obtained from A. sieversiana 5 ml/L extract treated plot was not significantly different from maximum tuber yield of 25.24 t/ha from plot treated with Refree, while minimum yield of 11.10 t/ha was obtained from the control plot and 23.99 t/ha yield was obtained from S. alopecuroides 5ml/l treated plot as shown (Table 2, Figure 2).

Assessment of potato yield loss indicated that white grub caused severe damage to tubers resulting in yield loss of up to 56.03%. Highest yield loss of 56.03% recorded control plots was not significantly different from 26.74% to 54.45% yield loss recorded in plots treated with 1 and 3 ml/l of S. alopecuroides extract and plots treated with 1, 3 and 5 ml/l of T. minuta, A. sieversiana, P. harmala extracts but significantly differed from lowest yield loss of 4.62% in plots treated with Refree, 26.74% and 8.25% from S. alopecuroides 3 and 5 ml/l and 35.64% yield loss from A. sieversiana 5 ml/l treated plot. However, yield loss of 26.74% and 35.64% recorded in plots treated with 3 ml/l S. alopecuroides and 5 ml/l A. sieversiana was not significantly different from highest yield loss of 56.03% from control and other plots. Results indicated that two applications of Refree at rate of 45kg/ha and S. alopecuroides 5 ml/l at rate of 100 l/ha significantly reduced the potato tuber yield loss to 4.62% and 8.27% from an economical yield loss of 56.03% caused by white grub attack during 2019.

White grub is an important agricultural root feeding pest widely distributed in different agro-climatic conditions of Pakistan and throughout the world. White grubs attack potato which is one of the four major staple foods having a significant contribution to national food consumption needs.

Results of present study revealed that S. alopecuroides fermented extract is most effective to manage white grub infestation reducing tuber damage under field conditions.

 

 

In concurrence with the results of present study, Yadava and Sharma (1995) reported that white grubs cause up to 70% damage to roots of commercial crops and one grub/m2 caused 80-100 per cent plant mortality. Similar damage rate in potato tubers varying from 8.5 to 75.05% especially in hilly regions of India reported by Chandel et al. (2015). Rani et al. (2009) found fermented extracts of Sapindus mukorossi, Chrysanthemum sp., Nerium oleander, Urtica diocia and Vitex negundo in indigenous cow urine @ 5 mL/L were equally effective and the per cent tuber damage on tuber number basis in these treatments varied from1.67-9.58% during 2007 and 5.08-7.46% during 2008 crop season. Similarly, the result of studies on effectiveness of fermented cattle urine (1:6) and natural products of Nicotiana tabacum, Azadirachta indica, Capsicum annum and Allium cepa in the management of barley aphid, Diuraphis noxia carried out in Ethiopia revealed that all the treatments were toxic to D. noxia and provided more than double increase in yield (Tesfaye and Gautam, 2003). Furthermore, Sahayaraj et al. (2011) reported more than 90% larval mortality in Spodoptera litura released onto castor leaves treated with 13, 18 and 23 days old fermented extracts of Calotropis gigantea and Pongamia pinnata leaves. In another similar study, Baloc and Bulong (2015) reported that fermented extracts of Azadirachta indica, Gliricidia sepium, Tagetes erecta and Tinospora rumphii were equally effective as the synthetic pesticides in terms of final plant height, average number of white flies reduced after spraying and average number of 28-spotted beetles reduced after spraying.

Conclusions and Recommendations

Results of this study revealed that white grubs cause considerable infestation and potato tuber yield loss. Fermented extract of S. alopecuroides a readily available plant has the potential insecticidal activity against white grub infestation on potato crop and can be used as a novel alternative to synthetic insecticides for eco-friendly management of white grub, a polyphagus soil insect pest damaging all sorts of field crops, forest trees and ornamental plants. Further studies should be focused on different fermentation techniques, formulations, phytotoxicity and application methods.

Acknowledgements

This investigation was carried out under financial assistance of Agricultural Linkages Program (ALP), PARC which is gratefully acknowledged. Authors are also thankful to Regional Coordinator, Economic Transformation Initiatives (ETI), Skardu for providing potato seed (var. Flamenco) to carry out the experiments.

Novelty Statement

Every years tons of chemical insecticides are being used to control white grub in spite of serious health and environmental risks. Our current study emphasizes the insecticidal potential of native plants for eco-friendly cheaper management of white grub infestation on potato and other field crops to reduce the use of harmful synthetic insecticides

Author’s Contribution

Muhammad Ayub, Syed Arif Hussain Rizvi and Shahid Hussain contributed to overall management of the research work, data analysis and writing of the article. Munir Hussain, Sher Ahmad, Muhammad Qasim, Muhammad Din, Muhammad Ishaque Mastoi and Tajudin gave technical inputs and proofread the paper.

Conflict of interest

The authors have declared no conflict of interest.

References

Agricultural Statistics of Pakistan, www.faostat.fao.org

Baloc H.A., P. Marissa and M.P. Bulong. 2015. Efficacy of fermented botanical plant extracts in the management of white flies and 28-Spotted beetles in tomato. Int. J. Sci. Res., 4(7): 2566-2569.

Chandel, R.S., M. Pathania, K.S. Verma, B. Bhatacharyya, S. Vashisth and V. Kumar. 2015. The ecology and control of potato white grubs of India. Potato Res., 58(2): 147–164. https://doi.org/10.1007/s11540-015-9295-3

Crocker, R.L., L.A. Rodriguez-del-Bosque, W.T. Nailon and X. Wei. 1996. Flight periods in Texas of three parasites (Diptera: Pyrgotidae) of adult Phyllophaga spp. (Coleoptera: Scarabaeidae) and egg production by Pyrgota undata. Southwestern Entomol., 21: 317-324.

Food and Agriculture Division, 2012. Agriculture statistics of Pakistan. Ministry of Food and Agric. and Cooperat. Econ. Wing, Govt. of Pakistan, Islamabad, pp. 230-290.

Hashmi, A.A. and Shafiullah. 2003. NASSD Background Paper: Agriculture and Food Security. IUCN Pakistan, Northern Areas Progamme, Gilgit.

Hostettmann, K.J. and W fender. 1997. The search for biological active secondary metabolites. J. Pestic. Sci., 51: 471-482. https://doi.org/10.1002/(SICI)1096-9063(199712)51:4<471::AID-PS662>3.0.CO;2-S

Ismail, M., F. Hussain and S. Ali. 2014. Botanical biopesticide and insect repellent from Tanacetum baltistanicum Endemic to Gilgit-Baltistan. J. Biodiv. Environ. Sci., 58(2): 128-135.

Mittal, I.C. and H.R. Pajni. 2000. New species belonging to (Coleoptera: scarabaeid: Melolonthidae) from India. J. Ent., 2: 85-88.

Musthak, A.T.M., G. Sharma, Y.S. Mathur and R.B.L. Gupta. 2013. Biosystematics of phytophagous Scarabaeidae- an Indian overview. In: (Eds.). Ind. Phytol., 2(5): 83-85.

Ouedraogo, M., A.M. Toe, T.Z. Ouedraogo and P.I. Guissou. 2011. Pesticides in burkina faso: Overview of the situation in a Sahalian African country. Pesticide in the Modern World: Pesticides uses and Management. Juneza Trdin 9, 51000 Rijeka, Croata. https://doi.org/10.5772/16507

Rafael, E., 2001. Botanical insecticides. www.ipmworld. umn.edu/silvia.html. Accessed on 12 October 2019.

Rani, P., Y. Thakur, A. Sharma, A. Sharma and V.K. Chandla. 2009. Biointensive Management of White Grubs with Botanical Extracts in Indigenous Cow (Hariana) Urine. Potato J., 36(1-2): 61-64.

Rausell, C.A., C. Martineze-Ramirez, Gracia-Robiess and M.D. Real. 2000. A binding site for Bacillus thuringiensis Cry 1Ab toxins lost during larval development in two forest pests. Appl. Environ. Microbiol., 66: 1553-1583. https://doi.org/10.1128/AEM.66.4.1553-1558.2000

Rizvi, S.A.H., S. Ling, F.F. Tian, J. Liu and X. Zeng. 2019. Interference mechanism of Sophora alopecuroides L. alkaloids extract on host finding and selection of the Asian citrus psyllid Diaphorina citri Kuwayama (Hemiptera: Psyllidae). Environ. Sci. Pollut. Res., 26(2): 1548-1557. https://doi.org/10.1007/s11356-018-3733-0

SAS Institute, Inc. 2010. SAS Note 40724, “Comparing covariance structures, testing covariance parameters using the COVTEST statement in PROC GLIMMIX.”. Cary, NC: SAS Institute Inc. Available at support.sas.com/kb/40/724.html.

Sahayaraj, K., P. Kombiah and D.S. Kumar. 2011. Evaluation of Insecticidal Activity of Fermented Plant Products on Spodoptera litura (Fab.). Indian J. Agric. Res., 45(1): 77- 82. www.arccjournals.com / indianjournals.com

Selmen, L., 1998. White Grubs-Polyphaga and other Species, Department of Entomology and Nematomology, University of Florida.

Shakur, M., F. Ullah, M. Naem, M. Amin, A.U.R. Saljoqi and M. Zamin. 2007. Effect of various insecticides for the control of potato cutworm (Agrotis ipsilon Huf, Noctuidae: Lepidoptera) at Kalam Swat. Sarhad J. Agric., 23(2): 423-426.

Silva, W.A.P.P.de, G.K. Manuweera and S.H.P.P. Karunaratne. 2008. Insecticidal activity of Euphorbia antiquorum L. latex and its preliminary chemical analysis. J. Natl. Sci. Found. Sri Lanka, 36(1): 15-23. https://doi.org/10.4038/jnsfsr.v36i1.129

Stoll, G., 2000. Natural plant protection in the tropics: Letting information come to life. 2nd edn. Magraf Verlag, Weikersheim. pp. 376.

Tesfaye, A. and R.D. Gautam. 2003. Traditional pest management practices and lesser exploited natural product in Ethiopia and India, appraisal and revalidation. Indian J. Trad. Knowl., 2: 189-201.

Yadava, C.P.S. and G.K. Sharma. 1995. Indian white grubs and their management. Technical Bulletin No. 2. Project Co-ordinating Centre, AlCRP on white grubs, Durgapura, Jaipur, pp. 26.