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?s5} . TTTE PHILIPPINEOURNALOFSCIENCE
CpfrfPlflllu.ni i"tRFl;f -Jmc 192 rssN 0031-7683 Vol. Ull No2
THE PHILIPPINBJOURNAL OF SCIE
ISOTATION AhID CHARACIERIZATION OTA MAI{NAIYOLYUC BACIERIIIM
lNS. Mendoza, B S. Cubol, E. G panerio and L. M. Jmon
ABSTRACT
tyhyryng aaivitv ond percentage dcgee of aegaaiaoi ii iirri *ri-drc. It also qrribited the wgnest eizyme"actiiry wnen grown in minerar sartsmedium containing either coconut residte oi to"urt bean mannan as soreEourceE. Th+fiect of pH and tempiraturc ronges for ortirity oy tncrudc enzyme were 5.0 a. 6.0 ana SO to Sf C, rcspectivety. paper chromuography analysis su8'ests morrnose as tne maiornyarotysi prokrriiini'i*a"enzyme.
INTRODUCITON
In coconut oroducing countries like the philippines, large vorume of coconutresidues is dbcharged ryty-fitoduciir uon- ary -a wet processes of oil extraction frommature coconut meat. The carbohydrate mmposition ofihe coco;;l endosperm is 74zomannose' ZLvo detfiose and smalr amounts of arabinose -a'lJ""tore (1,2). Thisindic/tes that the coconut mea'h "
ti;f;;;;;irt";""s which arehomopolysaccharides of D- .*opyr*ose residues.
The coconut meat or endosperm, however, has not been fully ut'ized for foodand feed despite its highprqtein conteni 1g b""uu." 6f i1s high crude fiber content. Itis therefore importanito utilize tt e Uy-plJa"cts from **fit;";; either as residueor as copra meal not only for food or feei but 4r9 to d*"6ffi;; its utirization intonore valuable products. one efficient method i, to ."*i'ror.iooorganisms whichwould hydrolpe the coconut resi{gg polysaccharid"r t";;; io "*a"
fiber contentand thereby increase oil extractabruf. iroiro""r, it seems "tt ""ti""
to convert the*:,9^.1911"" p_tgtr-T" .Ul niarl
", org*ir.
""p"Ur" oit ansforming thern'nnaF rnto some metabolizable carbohfdrates.-This p"pJ J*"iiu"s the identifica-
Vof,llll No.2
sctENCE At{D TECrr i . , .DEPARTMENT OF !I Y
!-liii-ii i,:iltllAIRIL-JI'NE 1992 rssN 0031-76E3
A b acterium is orued from dccomposing copra and identift ed as B acittusW6 selected the most ocdve Aanfrn'"riwrg;;;;;;;;ffi,
h&lrbl Tcchnolory Darclopmcnt Institute, Manila, philippines
I.l-
r.01
L02 Philippine loumal of Science
tion and characterization of an active nattnanolytic bacterium isolated from de,compos-ing copra.
MATERIAISANDMETHODS
Mateials
Coconut meat residue wirs prepared from frashly glated matured coconut ex-tracted of coconut nilk using a homogenizer. The residue was washed with acetonefollowed by an ether-alcohol (3:1) washing and then dried overnight at 80oC. Mannanfrom coconut residue was prepared according to the procedure of Mukherjee and Rao(a). Other substrates and chemicals were of reagent grade available commercially.
Media
The isolation medium contained the same mineral salts as used by Pablova andTin'yanova (5) and Jones and Ballou (6). Coconut meat residue (0,5Vo) was added assole carbon sour@. Platings were done on the same medi'm except that coconutresidue mrnnan was substituted for coconut residue.
Mehod of Isoluion of Mannanolytic Bocteia
Decomposing copra samples and adjacent soil mixture (2.5 d obtained from acoconut field in Nagcarlan, Laguna, Philippines were macerated and dispensed into 50ml isolation mediu- and incubated with shaking at room temperature for Z hours. Oneml of the suspension was transferred to new msdirrm and incubated under the sameconditions as above. This process was repeated five times to enrich for coconutresidue-utilizing bacteria, Dilutions of the supernatant were plated on mannan agarincubated at3fC and colonies showing areas of clearingwere observed.
Choacterization ond ifuntificuion ef the Organism
ffis rnannslslytic isolate was charactefizrdby standard biochemical and mor-phological techniques and Berge/s Manual was used for the identification.
Crudc Enryme Preparuion
Cell-free supernatants soalaining the enzyme was obtained after centrifugationof the 24-hr culture broth of the organism grown in liquid medium containing the mineralsalts mbcure wrth l% locust bean mannan.
EnrymeAssoy
Locust bean galactomennan was used as substrate. Reaction mfudure consistedof.O.SVo substrate in 0.1M acetate buffer, pH 55 and en4rme in a final volume of 1.0 nl.Incubation was carried out at 3fC for 10 min. Reducing sugars as mannose weremeasured by Nelson-Somogd method (7). A unit of en4me activity is defined as theamount of en4mae that releases l umole of mannose per minute.
t2t2 Mendazq et al.: Isolation andCltswterintiot 103of a Mannano$tic Bocterium
Grwbraic Assay of Enzyne Activity
The organisms were inoculated in flasks osn[aining the mineral salts medigm€rce'pt that peptone was omitted and yeast extract concentration was decreased from05 to0.tVo. r imilsd amount of coconut residue (0.17o) was added as sole carbon sourc€and incubated at 3fC for 1 to 2 weeks in a recipiocal shaker. The degree of solubiliza-tion of the coconut residue was measured gravimetrically using tle procedure ofIrmbeck and Colmer (8).
Paper Chromatography
Hydrolytic products from the reaction of the crude enrqe and locust beanmannan were analyzed by 2sssnding paper chromatogtaphy after passing through anionand cation exchange resins to ninimize streaking. The effluent wasevapoiated todrlmess, resuspended in 1.0 ml water and approximately 20 ul were spottJd on filterpaper (lbyo Filter Paper No. 50). The chromatogram was developed lor 24 hrs. withbltanol-pyridine-water (6:4:3, by volume). Reducing sugars were detected after afterdipping in silver nitrate-acetone solution.
RESTJLTS AND DISCUSSION
After a series of repeated enrichments and platines, seven out of 46 bacterialcolonies thatdevelopedin the preliminary scr'esning showed relatively high mannaaassactivitybased on size of clearing zones formed on nuuuran agar. Attempl was made toselect the best strain capable of utilizing mannan and results are shown in Table 1.Coconut meat residue was easily digested by streins No.39 and No.2. It seems that theP_hfsicat treatment (ball milling) subjected to the coconut residue could be enough inaltering the structures of the coconut polpaccharide fibers for it to be susceptible toen4rmatic degradation. This suggests the potentiality of the substrate consideringfurther that it is a waste product for SCP production.
Strain No. 39 also excreted the highest mannanase activity in the medium wheneither coconut residue or locust [es1 6ann31 was used as carbon souroe (Thble 2).Th's, it is apparent that the enzJ@e is inducible as both carbon sources consist largeiyof (l4)B-linked mannose residues. Although the orgenis6 grew well in the liquiimediurn without the carbon sources, insigpificant anount of mannanase was produced.
The selected strain of mannan-utilizing orgenisn is a small, motile, aerobigspue-forming gram-positive, rod-shaped bacterium. Its morphological and biochen-ical characteristics shoum in Thble 3 are identical with that of Bicillus subtilis describedin Bergp/s Manual.
Fgure 1 shows the effect of pH and temperaturg on mannanase activity of theisolated strain- F" p}I and temperature ranges of 5 tcj 6 and 50 to 5fq respectively*Ere n€c€ssary for optimp6 activity.
Our chromatographyof the hydrolpis products after}4.fu, incubation of locustfop6l mrnnan with the crude en4me showed that the basic product is mannose. In
+
104 Philippine loumal of Science t992
addition to mannose, faint spot overlapping that of mannose was detected. This
compound was not identified due to inavailability of higher mannooligosaccharides as
matlets. The results however indicate that the crude enzyme had substantial mannanase
activity and that the hydrolysis attack results in production of mannooligosaccharideswhich are further degraded to mannose thereby being metabolizcd by the cell.
REFERENCF,s
Hagenmaier, R.D., M. Glissendorf and K. F, Mattil. 1976.The residue from aqueousextraction of fresh coconuts: An Analysis. Phil. J. Coconut Studies. | (l):37-4L.
Balasubramanian, K. 1976. Polysaccharides of the kernel of maturing and matured
coconuts. J. Food Sci.41: L370-L373.
Hagenmaier, R.D. 1977. Coconut aqueous processing. San Carlos Publications, CebuCity.
Mukherjee, A., and C. Y. Rao. 1962. A mannan from the kernel of Coconut (Cocos
nucifera). J. Indian Chem. Soc.39: 687
Pavlova, I. and N. Tin'yanova. 1981. Isolation and characterization of microorganismswith mannanase activity. Appl. Biochem. Microbiol. L6: 427
Jones, G. H., and C. E. Ballou. 1969. Studies on the structure of yeast mannan. I.Purification and some properties of an alpha-mannosidase from an Arthrobacterspccie. J. Biol. Bhem. 't44: t043.
Somog;li, M. 1945. A new reagent for the determination of sugars. J. Biol. Chem. 160:61.
Lembeck, w. J. and A. R. Colmer. 1967. Effect of herbicides on cellulosedccompositionbySporocytophaga mlxococcoides Appl. Microbiol. 15: 300-303.
5
t2l2 Menfuzg a. al.: Isolation md Characterintion . 105of aMannanoW Bactertum
h
*Degree of substrate digestton rras measuredgravinetrlcally after the lsolatesr were lnoculatedtnto 50 ml nLneral ealts media containing 50 mgcoconut resldue as sole sarbon source, and lncubatedat 37oC for 5 to 7 days.
Table 1. Degradatton of coconut residue by nannanolytLc bacterLa
Strain No.rn i t ia l t { t .
n9
Degree of Digestion*
Final l{ t .n9
Solubil-izationt
1 8
9
2 0
1 1
1 4
39
2
5 1 . 5
5 0 . 7
5 1 . 4
4 9 . 8
49 .2
5 2 . 0
5 1 . 2
2 6 . 6
1 7 . 5
2 4 . 3
2 5 . 8
2 4 . 2
1 3 . 1
1 7 . 5
4 8 . 4
6 s . 5
52 .7
48 .2
5 0 . 8
7 4 . 8
5 s . 8
b
.LWz106 Philippine lountal of Science
*The crude enzyme obtained from the 24-hr growthof the isolates in the l iquid medium containing 1tof the carbon sources each (coconut resl.due andlocust bean mannan) was assayed for mannanaseacttvity as described in the Materials and Methods.
il Table 2. Irlairnanase activities of the mannanolytic bacteria
St ra in No.
Enzyme Activity*
Coconut Residueunit.s/rnl
Locust Bean Mannanun i ts /ml
1 8
9
2 0
1 1
1 4
3 9
2
0 . 1 3
0 . 2 5
0 . 1 8
0 . 1 8
0 . 1 9
2 . 1 8
1 . 7
0 . 3 9
0 . 6 9
0 . 2 3
0 . 2 1
0 . 2 8
1 . 7 7
0 . 9 8
1
t2t2 Mendozq et. al.: Isolation and Choncterizationof a Mannanolytic Bacteium
to7
i.!Table 3. ttorphological and Biochenical properties of
Bacillus subtilis and lltannanolytic Bacterium
Property
Morphological characteri stlcs B . sub t i l i s Isolate
ShapeS ize (un )SporesSwoIIen sporanglumMot i l i tyGram stain
Biochemical characteristics
GlucoseLactoseMaltoseSucroseArabinoseXyloseMannitolStarch hydrolysisCasein hydrolysisNitrate reductionIndole productlonOxygen requirerrrentCatalase testUt l l izat ion of c i t rateGrowth at pH 5.6Vogea Proskauer test zMethyl red test
irregular0 . 6 - 0 . 8 x 1
i
rod. 3
+
ActdAcidAcld
AcidAcidAcid
+++
+aerobic
+++++
irregular0 . 8 - 1 . 3
?
++
Ac idAcidAcidAcidAcidAcidAcid
++++
aerobic+++++
rod
(
108 Philippine loun@ffiwice
Temperature (oc)
6 ? 8
LW2
A 1 Cbf
+ ) .
.F{
isn(0
o.F{
+)(u
8zs
F ig . 1 .
pH
The effect of temperature (l) anO ptt (O) on the
mannanolytic activity of strain f{o. 39
Vol.illl NoJ PHILIPPINE JOTIRNAL OF SCIENCE
UTILIZATION OF COCONUT HUSIC A COMPAR/$WEINVESTIGATION OF THE ION EXCHANGE PROPERTIES
OF COCONUT HUSKIIBER
PREM NAIH MAIM2 and JGSE H. SANT{NInstttute of Chemistry College of Arts and Sciencs
University of the Philippines, Loc Baftos College, Laguna
ABSTRACT
Mercury (I ) -arhanged cocorwt husk fiber was employed for hydtotysisof 2-methyl-3-butyn-2-ol to 3-hydraxy-? methyl-2-butanone in 71,58 and51.95Vo yield by CH-IN and CH40 mesh sins of caconut husk fiberrcspectively, comporuble to 74.09Eo yield by Dowq-S0 rusin. The cationachanp capacities of CH-100 and CH-40 mesh sizz of huskfiberwere foundto be 0.98 and 0,75 meqlg respectivety.
INTRODUCTION
The cation exchange properties of naturally existi"g cellulosic materials havebeen studied by various researchers (Masters, 1!}22; Ludtke, 1936; Mclean and Wooten,1939; Hayrnann and Ravinov, 19al) and they have shoum that incorporated cellulosicmaterials exhibit cation exchange properties.
Ion exchangers are used as catalytic supports in chemical reactions. Severalorganic reactions are catalyzed by acids, alkalies or metal ions. They are employeddirectly or indirectly by exchanging them on the spthetically available ion exchangers.Nenman (1953) studied the hydrolysis of terminal triple bond to ketonic form in theabsence of, directly using and by supporting mercury (II) ions on Dowex-50 resin showingthe advantage if indirectly using the support in terms of higb major product yield,minimum side reactions and less vigorous reaction conditions.
Hg*Z,Hg+2-Dowex-50-C = C-H
The advantage of indirectly using solid have been demonstrated by variousexamples of conversions of terminal triple bond to ketonic group (GelbarllgTl;Yamawaki and Ando, 1979:' Gree et. al., 1980; tansbury et. al., 1980; Schick, 1982;Maini, 1986; Mnini st. al., 1988). Therole of Hgtz and H + ion has been documented(Marc\ 198t. To date, no studywas carried out to determine the efficiency and capacityof coconut husk fiber, although coconut fibers has been utilizpd for purification of
2.Prcscnt Adrcss: P.N. Maini Consulting Chemist, PhilRice Pili Driric, UPLB C.ampus, C.ollcge, Lc Baiios,I-aguna
ot l
{ -CHt
/o
19f/2110 Philippine loumal of Science
water (Fraenkel,1974) and in removal of heavy metal from waste water (Latif, 1989),which contains up to 43.tAVo of cellulose to act as a cation exchanger thus promptingthis investigation.
MATERTAIS AIYD METHODS
L List of Reagents: 2-methyl-3-butyn-2-ol, Mercuric (II) oxide, Dowex-5O resin,Silica gef Alumina, Florisil, Cellulose, Celite, and Molecular Sieves.
il. The investigation was carried out in four steps:
o Preparation ofhusk fiber.
o Determination of the cation exchange capacity of the husk fiber mesh.
o Preparation of mercurated husk fiber and other synthetic andcommonly used solid supports.
o Hvdration of 2-methvl-3-butyn-2-ol by the direct and indirect use ofui+2 ion.
Preparation of husk fiber:
Mature brown husks were used to obtain the husk fiber. The fiber was separatedfrom the coir dust by mechanical means and washed several times with water. Theseparated fibers were sun-dried for 24 hours and then cut into shorter pieces.These were further ground into uCI and L00 mesh sizes and subsequently dried inan oven at95-100oc for20 hours.The oven-dried fibers were storedintightlyclosedbotles.
Determination of the cation exchange capacity of the husk fiber mesh:
The ion exchange capacities of the prepared CH-40 and CH-1"00 fibers weredetermined by the volumetric method of llelfferich (L%2).
Preparation of mercurated husk fiber and other synthetic and comnronly used solidsuppotts:
About 10 g each of the solid supports CH-40, CH-L00, Dowex-50 resin, silicagel,alumina, florisil, cellulose, celite, and molecular sieves were treated with acid andmercury (II) solutions according to the procedure of Newman (1953).
Hydration of 2-methyt-3-butyn-2-ol by the direct and indirect use of Hg+2 ion.
The 5 g of 2-methyl-3-butyn-2-ol in 30 ml of methanol - water mixture (2:1)containing 3-4 drops of concentrated sulfuric acid was stirred with 5 g of mercuratedhusk fibers at room temperature for 24 hours. The same method was used for the othersolid supports. The final mixture was filtered free from husk fibers (or other solidsuports). The filtrate was diluted with 20 ml of water extracted thrice with 20 ml aliquotsof chloroform. The combined chloroform extracts was washed thrice with 20 ml of water,and then dehvdrated with anhvdrous sodium carbonate. The chloroform was distilled
ll
l2l2 Maini and Santos: Utilizntion of Coconut HuskAComparative lnvestigation of the lon Exchange Prop.
off and products from various reactions were collected at L*-L4ZoC. The yield of theproduct obtained area on the basis of the reactant used. The reaction was carried outwith mercury (II) alone, without mercury, and with mercurated solid supports. Theproduct 3-hydrory-3-methyl-2-butanone was identified by its infrared and protonmagnetic spectra and 2,4-dinitrophenyl-hydrazone derivatives.
RESTJLTS AND DTSCUSSION
The cation exchange capacity of two fiber mesh sizes CH-40 and CH-1"00 wasdeterminsd to be 0.75 and 0.98 meq/g. The difference in the ion exchange capacities canbe e4plained by their different mesh sizes.In the preparation of mercury flI) exsfuangedmeshes, no physical or visual changes were observed (see Table 1).
The hydration reactions were carried out at ambient conditions to provideidentical reaction conditions, since somesynthetic supports are unstable at high temper-atures. The yields of the product 3-hydrory-3-rnethyl-2-butanone, with various reactionreagents are shown in Table 2. The husk fiber untreated or treated alone with acid ormercury (II) alone failed to effect the reaction given below, which evidently shows therole of solid support in the reaction.
111
CHr
C H g - C - C = C HI
OH
(gg*2, H*)cHr ol t
Using Hg (II) ion at 1M concentration only led to a poor yiel d, of 3.8%. Dowex-fl)resin showed 1[s highg5l yield ,74.09Vo owingto its knsqm high cation exchange capacityof 4.9 - 5.2 meq/g. Curiously, CH-100-Hg" mercurated husk fiber despite its muchlower exchange capacity gave a comparable yield of.7t.?8Vo showing that the husk fiberof CH- 100 mesh size is effective and efficient as an ion exchange support for thisreaction.
The product 3-hydroxy-3-methyl-2-butanone was easily identified due to IR signalat L730 cm-' indicating the presence of a carbonyl group (- C -) and by the disappear-ance of the PMR acetylenic peak at2.46 and appearance of an additional methyl signalat 2.26 for CH-40 and CH-100 mercurated husk fiber (see spectra I&II). Also, thel&dinitrophenyl hydrazone derivative of 3-hydroxy-}methyl-2-butanone from various ,reactions showed melting points in the same range as reported in literature (1,10 - 141$C)(see Table 3).
STJMMARY A}.{D RECOMMENDATIONS
This investigation confirmed that cellulosic materials can be used as cation-ex-changers even in their natural form as exemplified by coconut husk fiber. It was alsofound that particle mesh size affects the ion exchange capacity of the husk fiber.
I#t
Philippine loumal of Science
The successful conversion of the given acetylenic compounds by Hg(II) treatedcoconut husk fiber in comparable high yield, proved that coconut husk fiber can beutilized as a low capacity ion-exchanger and solid support for the catalytic hydration oftriple bonds.
However, in order to prove the general use of coconut husk fiber as a catalyticsupport material, or as a low capacity ion-exchanger, a detailed study of more terminaltriple bonded compounds and other classes of catalytic reactions is necessary.
ACKNO\ryLEDGMENI
The senior author wishes to thank the UPLB Administration for the financialsupport throughout the investigation and the KKP (Kapisanan ng mga Kimiko saPilipinas) for the thesis grant awarded to the junior author.
REFERENCES
FRAENKEL, R. J.. 1974. Evaluation of pilot water treatment using ine:rpensive 10 calmaterials as filter media for supplying drinking water to rural communities in thelower Mekong basin countries. Asian Inst. Technol., Bangkok, Thailand.
GELBARD ,G.l977.Anionic activation in polyner supported reactions; Nucleophilicsubstitution with anion-exchange resins. Synthesis. 113.
GREE, R., PARK H., and PAQUETTE, L.A.. 1980. Regio- and stereoselective 1,2Wagner-Meerwein shifts during trifluoroacetic acid catalyzed isomerization ofunsymmet r ica l l y subs t i tu ted t r i cyc lo [3 .2 .0 .V ' ' l hep tanes .
;102:4397-4403.
HELFFERICH, n lg62.lonExchange. McGraw-Hill Book, New York.
HAYMANN, E. and RAVINOV G.. 1941. The acid nature of cellulose II. The strengthof the cellulosic acid. J. Phy. Chem. 45: lL67-1I76.
LAIIE, P. 1989. Adsorption of copper II, zinc II and lead II by selected agriculturalwastes. Jaffar N Pertaiba. 12 (2) t9T2m.
I-ANSBURY, P. T, SERELIS, A. I(., HENGEVELD, J. 8., and HANGAUER, D.G.JR. 1980. Total spthesis of pseudoguanines - I. Preparation of bicyclo[5.3.0]decaneslmthons for damsinic acid and helenanolides. Tbtrahedron.36: nW'n10.
LUDTKE, M. 1935. Studies on acid properties of cellulose and the problem of oxidationprocesses on membrane substances. Biochem. 2.285:78'97.
MAIM, P. N.. 1984. 2,2-Disubstituted-1,4-diketones and their conversion to pyrrolederivatives. PhD Thesis. University of Hong Kong Hong Kong.
l$
ntA, Maini and Santos: Utilization of Cuonut HustcA Ll3Compuative Investigation of the lon Erchange prop.
MAIM, P. N., SAMMH, M.f" and I(ATRFZKY, A. R.. 1988. The spthesis andchemistry of azolenines' Part 8. The Paal-Knorr reaction wiih cyclic 2-(acylnethyl)-2-alkyl-1,3-diketones: Isolation of 1-ad-1H-pyrroles via rearrange-ment. J. Chem. Soc., krkin Thans.I: 161.
MARCH, J. 1985. Advanced organic chemistry.3rd edition. John wiley and sons.NewYork L346pp.
MASTERS' H. L922. Reactions of cellulose with sodium chloride and other neutral saltsolutions. J. Chem. 5u,., l2l: ?n?6.
McLEAN, D. A., and WOOTEN, L. A. 1939. Cation excharge in cellulosic materials.Ind. Eng. Chem. 31: 1138-1143.
NEWMAN' M. S. 1953. Reactions of acetylenic compounds by sulphonated polystyreneresins. J. Am. Chem. Soc. Ull:4740.
SCHICK H., SCI{WARZ, S., FINGER, A., and SCHWARZ, S. 1992. ZZ-Dis-ubstituierte Clclopentan-1,3-Dione-2. Untersuchung Der Regioselektivitat vonAlkylierungs. Tbtrahedron. 38:. L279 - L?A3.
YAMAWAKI, J.; and ANDO, T.. tg7g. Potassium fluoride on inorganic solid support.Chemistry lgetters: 7 5-7 58.
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Maini and Santos: Utilintion of Coconut HuslcACunpafatiw Invv*iiuion of the Ion F,xchaige prop.
115
nSCILiP SUFFOFIT FEF: ' :ENf YIEL.D { l '1 ,* rX, } r:[:f: { l.tH{;,/$ ]
trH* t t10-Ftg+I
rF.l-.,,+ (1..-. l.{g "l"I
r:l-.t-. 10rl-.H+
f,l-l-."4cl-.f{'
fit{* 100
r:l-{*40
Dr:rWeri*50
5 i l i c a g e l
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t le l l u l , rse
tlel. i te
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o . 7 5
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* t lat iun exclrange ' :apa' : i t ie i ls i (r :E.f f ) werer cletarr. in ined, r :Hr: f r , rc , t l re r mater ia ls r i ted i r - r tab le f i r .o wt l l l l , :n , : :wn
7 1 . t 8
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118 Philippine loumal of Science L992
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Yol.121No2 PHILIPPINE JOURNAL OF SCIENCE
COIJONIZATION AND ABUNDAI{CE OFARTHROPODS oNcucuMBER (cucuMrs sATrvtJS L) ATVARTOUS STAGES OF
PI-A.NT GROWTTI
EVELYN A. BERGONIA3 and CLEOMS R. CERVANCIA4
ABSTRACT
Insect pests, pollinatorc, and other arthropod associated with cucumber(uPL cu-6) were monitored throughout their growing stages Irom Matth toMqt 1989 at the University of the philWines , Los Bafios, Laguna
Colonization by insect pests, pollinatorc, parasites, predaton and tour-bts associated with cucumber was generally influenced by plant age. Ingeneral the afthropod population density increased from seedling to vegeta-tive and reproductive plant stages and decreased as the crop senesced-
The colonization and temporal abundance pattems were discussed.
INTRODUCTION
The increasing demand for picking cucumber provides a good opportunity forlocal growers to expand cultivation. In spite of the crop's adaptability and fast growingmarket, cucumber production has not fully established because of inadequate supply ofse-eds (Gregorio, 1989). Low yields are usually attributed to insect pests, diiiases,adverse climate and inappropriate cultural practices. Cervancia and Iiergonia (1991jreported that inadequate pollination reduced fruit and seed set.
Many insects and other arthropods are associated with cucumber from seedlingto maturity. Thrip infestations caused considerable yield reduction (Welter et al. 1990]Rosenheim et al. 1990). Aphid populations were monitored by Basky and Nasser (19s9)where.three species (Aphis goospil Glover, Aphis craccivora Kock and Myzus persicaeSulzer) were found vectors ofvirus causing diseases.
Screening for insect resistance has been done. Eight of 1160 cucumber lines$ow9d resistance to picle worn Diaphania nitidalis Stoll (Wehner et al. 1985). Basky(19861 noted the importance of natural enemies in regulating aphid populations inHungary and cautioned that chemical methods are dangerous to piedators.
I\donitoring the colonization and temporal abundance of arthropods will providebasal data for development of alternative management program for cucumber produc-
3
nDepartment of Entomologr ,University of the philippines, College, I:guna
Jnstitutc of Biological Sciences, University of the philippines, College, l_agona
lfr119
L20 Philippine loumal of Science IWz
tion. Such program should include regulation of pest population densities while con-
serving natural enemies, pr6llin31615 and tourists'
MAIERIAIS A}.ID METHODS
Land Preparation, layout ond planting
A 600 m2 area in the Central Experiment Station, University of the Philippines
at Los Banos was thoroughly harrowed and furrowed at a depth of 30 cmn. Seeds of a
local picking cucumber variety, UPL Cu-6, were sown at a distance of 75 cm between
rows and l0 cm between hills. Ttellises made of wooden poles and straw were installed
to support the growing plants. All the cultural practices recommended by Deanon and
Angat (1984) were followed.
Observations were done from March to May 1'989.
Colonization and abundonce
Arthropods were monitored weekly during the growing stages of the crop.
Samplings weie mostly done early in the morning to avoid diurnal changes in species
visiUitityl nor floral visitors, observations were done during anthesis. Sampling was done
by the following methods:
1. Sweep net sampling. The insect sweep net (38 cm diameter) was used to
sample -ote -obile inseCts. A total of 10 sweeps were taken. Because of the presence
of wboden trellises within the plots, brush strokes were done in between rows. The net
contents were etherized and brought to the laboratory for identification.
2. TlapsJ Sticky broad traps (12 x 32 cm) painted yelfow and red were placed
horizontally alove crop canopy. Four traps (2 yellow and 2 red) were placed equidistant
in each ploi. Ordinary grease *"ur used as sticker. This was spread eueory on ethylene
plastic iut just exactftie size of the board and fitted to the trap board. The plastic on
ih" ttup. o""re changed every after three days. Kerosene was used as solvent to separate
the insect from grease.
3. Per plant sampling. Five randomly chosen test plants, each from the 5 rows
within each cucumber plot were examined for foliar pests. Individual oounts were
recorded from the lrst jfu[y opened leaves of each plant sample. The arthropods were
grouped into three: 1) pesis; 1) natural enemies and tourists; and 3) pollinators and
iower visitors. Tourists are non-predatory species which have no permanent association
with vegetation (Moran and Southwood, 1982).
RESI.]LTS AND DISCUSSION
Pests
A total of 1L species colonized the crop (Fig. 1). The earlist colonists, thrips(Thrips palmi Kirkaldi) and squash beetles (Aulacophora similis Olivier) were observed
il
l2l2 Berpnia and cemancia: colonization andAbundsnce tzLof Arthrcpods on Cucumber (Cucumis Satiws L.)
at seven days after emergence (DAE). A. similis mdT palmaz peaked at 14 and42DAEand27 to 28 DAE, respectively.
At 14 DAE, tw9 mitg-spgcies (Amblyseius sp. and Tbtanychus sp.), melon aphids(Aphis gossypii Glover) and leaf folder larvae (Diiphania indiu Saundin)*"r" udd"d.All populations peaked at this stage and persisted until crop maturity.
Other species like 28-spotted beetle (Epilachna philippinensis Dieke), ladybirdbeetle (Micraspis uocea Mulsant, mealybus (Fenista sp) a"d katydid (phaneripterafurcifera Stal) werc not,numerous and present only at certain stages of plant grottn.Theywere not observed to cause economic damage to the crop.
Natural enemies and touists
-- Jh" species of parasites, predators and tourist which colonized the crop werelisted in Table 1. Some important crop pests could have come from adjaceoi n"tA.planted !9 gto, sugarcane, bitter gourd and legumes. The most numeious speciescollected belonged to Diptera (Culicidae, Dolichopodidae and Muscidae), followed byColeoptera (Chrysomelidae) and Hymenoptera (Braconi<lae and Chalcididae). Otherspecies observed were scarce.
Pollinators and flower visiton
Seven species were observed foraging on the flowers (Fig. 2). These wereEumenes sp., Formicidae, Xylocopa sp., catochrysops cnejus Fabicius, chrosodeiischalcites Espeq, Diaphania indica saunderc and pamara sp. The populatibns were lowwith peaks at 21 and 28 DAE. Insect pests and mites, though reprlsented only by 11specieswere the most numerous (90%).This isbecause of the highpopulation densitiesof mites, aphids and thrips. Their small size is compensated by hlgh relproduction rates.It is also evident that these species have a wide habitat ratrge because they were presentfrom seedling stage to maturity. Thrips and aphids preferied younger piants as shownin their population peaks (21 and 28 DAE)'M1te populations,
-tto*"iuer, p"aked at both
early (14 DAE) and late (42 DAE) stages. This shows that mites are'liss affected byplant age and structure.
The most diverse group was the natural enemy and tourists (42 species), althoughtheir population density was low (10%). No relationship, however, coutd be establishJdbetrveen the populations of pests and natural enemies. It was noted, however, that thepredatory coccinellid (Menochilus semraculatus) colonized the crop simultaneouslywith their host, A gosslpii.,Predatory species are generalists that they prey on touristsas well. It was also difficult to separate the true predatory anO paraiitic species foridentifications up to-species level were not available. Most species were ideniilied onlyup to families. The interactions, then, between prey and pre-dator were not consideredin this study.
- -Therewereonlyfewspeciesof flowervisitors (lvo\. Thepopulationsweremostabllndnal at the peak of flowering stages (21 and 28 DAE). Speciejvisit the flowers forpollen, nectar or oviposition sites. using Faegri and van der iijl,s engD criterion, only
&k
rn. Philippine lanmal of Science
Eumenes sp. andXylocopasp. were biotic pollinators. These species visited the blossomregularly and their visits constituted a regular part of their life activity. Moreover, theydid not cause any damage to the flowers. Their foraging synchronized with anthesis.The absence of other bee species could be due to the destruction of their nesting sitesespecially in a developed agroecosystem.
Arthropod abundance sigaificantlyvaried with season (Table 2).
Generally, arthropod populations were low at early stages of plant growt\increased with season, reaching peaks at mid vegetative and reproductive stages anddecreased as crop senesced. It shows that plant stage and structure influence coloniza-tion and temporal abundance. At early plant growth stages, habitat is limited allowingonly for herbivores and species seeking shelter and oviposition sites. As the plant grows,habitat also increases providing more food and shelter to the species. At reproductivestages, additional food sources are provided" like nectar and pollen grains.
The species present throughout the season were relatively few. This maybe dueto the absence of natural vegotation near the site which can serve as potential source ofcolonists. Removal of uncultivated strips alters the succession of nectar and pollenyielding flowers foraged by pollinators and other species. Pesticide usage in the exper-imental area could have affected populations ofarthropods specially the top predators.
ACKNOWLEDGEMENT
The International Foundation for Science is acknowledged for supporting theresearch.
m2 Beryonia and Cenancia: Colonization and Abundanceof tuthropods on Cucumber (Cucumis Satiws L.)
123
REFERENCES
Basky, z. L9t5. Natural elelxes of aphids living on seed cucumber. In: Ecolog5r ofAphidophaga. Proc. of the 2nd symposium held at Zvkovske Podhradi.: Zn-{lt.
Basky, Z. and M.A. Nasser. 1989. The activity of virus vector aphids on cucumbers.Agric. Ecosyst. Environ. ?5Q): 337 -"a2.
Cervancia, C.R. and E.A. Bergonia. 199L. Insect pollination of cucumber (Cucumissativus L.) in the Philippines. Acta Horticulturae 288: ng-ZgL
Deanon, J.R. and E.A. Angat. 1984. Pamamaraan ng pagtatanim ng pipinong may tulos.Information Development and Dissemination Section. Depiitment of Horticul-ture. University of the Philippines-Los Banos.
Faegri, IC and L. van.der PijI. 1979. The principles of pollination ecolog5r. 3rd ed.Pergamon Press (Oford). zdAp.
Gregorio, G'L. 1989. Effect of defoliation, fruit maturity and position on seed yield,quality and storage potential of cucumber (Cucumis sativus L.). Ph.D. Dissertation.University of the Philippines-Los Banos.
l{oran, vc. and TR.E. Southwood. 1982. The guild composition of arthropod commu-nities in trees. J. Anim. Ecol.51: ?Ag-ffi.
Rosenheim, J.A., s.c. welter, M.w. Johnson, L.R. Gasukuma-minuto and R.EL. Mau.190. Direct feeding damage on cucumber by mixed species infestations of twoSipt, Thripspalmi and Frankliniella occidentatis lfnyianoptera: Thripidae). J.Econ. Ent. $(a): $19-1525.
wehner, Tc., K.D. Elsey, G.G. Kennedy. 1985. screening for cucumber antibiosis topickleworm. Hortscience. 2nG): LLLT-LLLI.
welter, s.c., J.A. Rosenheim, M.w. Johnson, R.EL. Mau and L.R. Gasukuma-minuto.1990. Effects of Thrips palmiand western flower thrips (Thysanoptera: Thripidae)on the yield, growth and carbon allocation pattern in cucumbeis. J. Econ. En[.83(5):2092-2L06.
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tu Philippinc lunrtal ol Science - twz
Table 1. lfunber of gredators, parasltee anil tourists at clllferentglant gronth stages.
}RTHROPOD SPECIESDATS MTER SEEDLING E}IERGENCB
? 1 { 2 L 2 8 3 5 1 2
t
AranaeLycosa sP.oxyopes sP.Unldent l f ied sP.
ColeopteraChrysomlldae
Casslda circundatalfonolePta Difasciata
Cocclnell idaellenochi Ius sexnaculatus
DcrrnestldaeTenebr ioniclae
DlpteraAgronYzldaeCectdonYlilaeChlrononlilaeCul lc lilaeDollchogoilldaeDrosophllldaeEghldrldae}{usclalae
llurca aP.Phorldae0t l t t ldaePaychodlileeTlpul ldae
llemlgtcraDysdercus ctngulaturlllr ltlaelletara vlrldulaCorsttlae
000
0 0 0 0 1 20 0 0 0 3 30 0 3 1 0
18182
883117
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3 1 { 8 3 3 4 31 3 2 L 0 05 0 3 2 1 0
0 1 6 1 0 1 23 0 0 0 0 21 1 1 1 0 2 ' . l
0I10
0200
1526I
7 45122t2298
2L0I
26
32 17 11 19l { 16 11 13{ 0 0 0
t5 38 s { 7266 3{ 15 151 1 1 3 2 L 113 11 19 1118 1? 28 1 l3 8 1 { 2 9 90 0 0 00 0 0 00 0 0 12 0 0 0
0 1 0 {2 0 0 00 0 0 01 0 0 0
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ARTIIROPOD 3PECIBSDAYS IFIBR SEBDIING EIiIER@ICB
? 1 1 2 L 2 8 3 5 4 2
9 1 5 0 1 0io tooo2 0 3 0 0 00 0 0 0 0 00 0 0 0 1 1
HomopteraClcatle I l ldae
t{ePiotettlx sP.DelphacltlaeFulgorldaeRlcanla sPeculun
HyrunoPteraBraconlitae
APanteles sP.ChalcldlttaeEncYrtldaeIchneunonldaePlatYgastertdaePteronaIItlae
OilonataCoenagrlonldlaeLlbellulltlae
0rthoptcraAcrltlldaeGrYllldacTcttlEonl ltlae
r 2 0 0 22 5 2 2 1 31 1 1 1 2 80 0 1 0 00 3 8 5 12 3 0 0 11 0 2 0 0
100000000101
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19v2t?f. Philippine lanmal of Science -
Table 2. llean nunDer of dlfferent arthropoils collected at clifferentdays after seedllng emtgence of cucunber (lfarch-llay, 1989)a
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DAYS IFTER SBEDIINGB}IBRGEIICE (DAE)
ARTHROPOD SPECIESPolltnators tourlsts TOTN.bInsect
Pests
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t2l2 Bergonia and Cenancia: Colonization and Abundanceof Arthropods on Cucumber (Cuatmis Sativus L.)
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Figure l. Colonlzotion ond femporol obundonce polterns of mlt€s ond lnsccf pesfs collectcd incucumber ( UPL Cu-6 ) from Morch to Moy 1989.
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Flcurc 2. Colonlzoflon ond ?cmporol obundoncc Poltcrn!- of polllnotors ondrother flowcr vlcltorr ofcucumbrr from Mcrch to Moy 1989
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