MARDI Res. Bull., (1985) 13,2: (166-171)

PATHOGENICITY OF PENICILLIUM FUNICULOSAM STRAINS FROMPINEAPPLE FRUIT BLEMISHES

W.H. L IM*

Keywords: Penicillium funiculosum, Pineapple, Pathogenicity studies.

RINGKASAN

Kepatogenan enam strain Penicillium funiculosum dan satu sttain Fusarium moniliforme yang

diasingkan dar ipada pelbagai b int ik buah nanas te lah diu j i ke atas buah nanas (cv. Masmerah) dan

tanda-tanda yang dihasi lkan te lah juga din i la ikan. Jenis stra in yang berbeza menyebabkan bint ik yang

ber la inan. Penyaki t mata dalam ( leathcry pocket) adalah disebabkan oleh dua stra in P. funiculosumiai tu Sl dan 55 (media t idak berwarna merah). Penyaki t mata hi tam ( f ru i t let core rot) d idapat i

d isebabkan oleh stra in 56 (media berwarna merah) manakala penyaki t b int ik gabus (cork spot) pula

disebabkan oleh stra in 54, sejenis stra in P. funiculosum yang menghasi lkan 'coremia' . Fusar ium

moni l i forme hanya menyebabkan tanda-tanda penyaki t mata hi tam yang terhad. Dua stra in ber la inanyang juga berwarna merah. ia i tu 52 dan 53 t idak menjangki t i buah nanas. Stra in-stra in ber la inan

didapati berbeza-beza di dalam kawasan-kawasan penjangkitan mereka.

INTRODUCTION

Six morphological strains ofPenicillium funiculosum isolated frompineapple fruit blemishes were distinguishedin Peninsular Malaysia (Lt t r l , 1983). InHawaii, three strains were distinguished,but only one, a non-pigmented strain wasable to induce leathery pocket disease.fruit let core rot and interfruit let corking(Lrv and RoHRsecH, 1980). RoURBACHand PnErrrER. (1976) demonstrated thatinfection by P. funiculosum occurredprimarily at four to seven weeks afterchemical forcing i.e. during earlyinflorescence development. Investigationsby the author on the local P. funiculosumisolates confirmed that for leathery pocketdisease, infection occurred at two to fiveweeks after forcing i.e. at earlyinflorescence formation.

Fusarium moniliforme has beenassociated with fruit let core rot in Hawaiiand other countries (RoHnencH andPnernnen, 1976).

In the present investigation, thepathogenicity of six strains of P.

funiculosum and one of F. moniliforme wasstudied in relation to the stage of develop-

ment of the pineapple inflorescence.

MATERIALS AND METHODS

Four ser ies of exper iments involv ingdifferent morpho-strains of P. funiculosum(LIv, 1983) and one strain of F. monil iformewere conducted over a period of threeyears. The strains of P. funiculosum weredesignated numbers, v iz . 51, 52, 53, 54, 55and 56 and were represented by isolatesB E l 1 , L P l , B E 9 , L P 1 1 , C S 3 0 a n d B E 3 0respectively. In all the experimenls, Ananascomosus cv. Masmerah was used as the testp lant as i t was the most suscept ib le cul t ivar( L r v , 1 9 8 1 ) .

The double row planting pattern wasadopted, spaced at 0.91 m (between doublerows), 0.61 m (wi th in a double row) and 0.3m (along a row). The plants were induced orforced to flower at 11 months after planting.

Two-week-old cultures (originatingfrom single spores) grown on potatodextrose agar at 30'C + 2oC were usedfor inoculat ion. The spore suspension wasprepared by using disti l led water containing0.057o Tween 40, d i lu ted to about I x 107spores/ml, as determined with a haemocyto-meter. Five mill i l i tres of the spore

*Frui t Research Div is ion, MARDI, Jalan Kebun, Kelang. Selangor, West Malaysra

r66

suspension was spray-inoculated into theheart of the plant using a 250-ml mistsprayer.

For disease assessment, the fruits weretaken to the laboratory and shelled. Eachfruit was then counted for the number ofhealthy and infected fruit lets (eyes) showingleathery pocket, cork spot and black eye(fruit let core rot) symptoms. Diseaseseverity was expressed as the mean numberof infected eyes per fruit.

Re-isolations were made from infectedfruitlets for confirmation of pathogenicity ofthe various strains.

Experiment I - Comparison of Strains Sl,53 and 34

Strains 51, 53 and 54 were comparedtogether with control (sprayed with steri ledisti l led water containing0.057a Tween 40),in a Randomized Complete Blockexperiment with four replicates. Ten plantswere treated per plot. The plants werespray-inoculated at 35 (early inflorescence),55 (early anthesis) and 65 (mid-anthesis)days after forcing (d.a.f.). The plants wereforced with carbide (6 g/l itre). All the fruitswere harvested for disease assessment twomonths after the last inoculation.

Experiment 2 - Confirmation of InfectionSites of Strains Sl and 54

The infection sites of 51 and 54 wereinvestigated. The treatments were (i) spray-inoculated into the 'heart' at 35 days afterforcing (with NAA pil ls) at the rate of 10 mspore suspension/plant, (i i) 0.3 ml sporesuspension introduced into each openflower and (i i i) spore suspension applied ondecaying flowers (petals) with a brush (ca.0.2 ml/flower). For treatments (i i) and (i i i)every treated fruitlet was marked with a dabof paint on the bract. The fruits wereharvested at two-eye ripe stage and the typeof symptoms obtained and disease severitywere recorded. Each treatment had a set ofcontrol where steri le disti l led water (plus0.05Vo Tween 40) was used. Fifteen fruitsselected at random, were sampled per

treatment. The data were analvsed bv theChi square test.

Experiment 3 - Comparison of Strains Sl,52, 53 and 54

In th is exper iment , s t ra ins S1, 52, 53and 54 were compared using the Ran-domized Complete Block Design with fourreplicates. There were 20 plants per plot.NAA pil ls were used to induce flowering.The 'heart' of the plant was spray-inoculated with the respective sporesuspensions at 30 and 40 days after treat-ment. About 15 to 20 fruits were harvestedper plot at maturity and assessed for fruitblemishes as described earlier.

Experiment 4 - Comparison of P.funiculosum Strains Sl, 52, 53, 55 and 56with .F'. moniliforme and their lnfection Sites

For this experiment, no statisticaldesign was adopted because of insufficientnumber of plants. However, the main plottreatments consisting of the five strains of P.

funiculosum, F. moniliforme and control,were randomized. Each main plot consistedof two sub-plots, viz. inoculation beforeanthesis (30 and 35 d.a. f . ) and inoculat ionduring anthesis (mid-flowering), which wererandomized. In short, the trial followed asplit plot design but without replication.Each main plot was separated by a doublerow of pineapple plants which acted asguard row.

The plants were induced to flowerusing ethephon at 250 ppm and 2Vo urea.During inoculation at anthesis, a polybagguard was used to minimize spore drift.Twenty fruits per plot were harvested attwo-eye ripe and assessed in the usualmanner for the various fruit let blemishes.

RESULTS AND DISCUSSION

Experiment I - Comparison of three P.funiculosum Strains

The three strains of P. funiculosum,51, 53 and 54, inoculated at early inflores-cence development, anthesis and post-

t67

anthesis showed significant differences insymptom expression (Figure I andTable I).Stra in 51 ( iso late BE1l) which was non-pigmented was pathogenic causing onlyIeathery pocket symptoms, strain 53 (isolateBE9) which was red-pigmented was non-pathogenic whi le s t ra in 54 ( iso late Lpl l )which was red-pigmented and producedcoremia was pathogenic but caused onlycork spot symptoms. As the same inflores-cence was inoculated at different stages ofgrowth, the site of infection for the threestrains could not be ascertained althoushear l ier s tudies in Hawai i had shown that forleathery pocket the infection site is on theyoung emerging inflorescence prior tc.ranthesis (RoHnnncH and PpElr rEH. 1976).

Figure I. Pineapple J'ruits showing differentsymptom expiession after inoculation withisolates LP I I (54), BEI I (St ) and BE9 (531

wu.\ non-pathogenic.

Experiment 2 - Infection Sites of Strains Sland 54

For th is exper iment the emerginginflorescence, the open flowers and thewi l ted f lowers were inoculated separate lvwi th s t ra ins Sl and 54, to determine thei rin fect ion s i tes. This conf i rmed ear l ier resul ts(L rv , 1982 ) , t ha t f o r S l ( i so la te BE I ) .infection is at the early inflorescence stagewhi le for 54 ( iso late LPI l ) , in fect ionoccurred for all the three stages studied

Table 1. Pathogenic comparison of threeP. funiculosum strains, Sl, 53 and 54

' l ' rcatmcnt Mean no. infcctcd cycs/ f ru i t " '

[-cathe'rv pockct Cork spot

S l ( l s o l a t c B E l l )

53 ( l so l a t c BE9 )

54 ( l so l a te . LP I l )

Contnrl

t i .3 a

l . ( ) b

t . 9 b( ) .7 b

0 .0 b

0 . t b

I tl.-1 a

t . 0 b

S . E . ( m c a n ) 0.9

" ' Mcans in columns fo lkrwcd by thc samc lct tcr arcnot s igni f icant ly d i t tbrcnt wi th p>0.05 as dctcr-m incd by DMR t cs t .

N.B. No black cyc (or f ru i t lc t corc rot) svmptom wasobscrvcd on thc f ru i ts samJr lcd.

(Table 2). The study also confirmed that Slcaused only leathery pocket whi le 54, coulc lonly cause cork spct t . There was no s igni -ficant difference in black t-.ye diseascbetween the twc'l isolates and cctntrol.

Experiment 3 - Pathogenic Comparison ofStrains Sl, 52, 53 and 54

The resul ts of th is exper iment con-f i rmed that of the four s t ra ins tested only 54could induce s igni f icant cork spotsymptoms. Stra in Sl produced thc h ighcstd isease sever i ty score for leathcry pockct(7.3 in fected eyes/ f ru i t ) a l though i t was nots igni f icant ly d i f ferent f rom thc othcr s t ra ins(Table 3/. This may in part bc cluc trrd i lu t ion of the spr t re krad by the ra in tharfe l l two days in succession ( t t .3 and 9.0 mnr/day respect ive ly) af ter thc l ' i rs t inoculat ion

This exper iment fur ther c<ln l ' i rmcclthat cork spot d isease was pr imar i ly causcclby 54. There were no significant dif l 'ercnccsin b lack eye sever i ty between the l i turst ra ins tested and the b lank contro l .

Experiment 4 - Comparison of p.funiculosum Strains Sl, 52, 53, 55 and 56with F. moniliforme, and their InfectionSites

In th is exper iment , two newly iso latedstra ins of P. J i ln i< 'u losum,designated 55 and

0 . 9

168

Table 2. Comparison of Strains 51 and 54 with regard to types of symptoms and infection site

Stage of inoculat ion

Symptom/strain Emerging inflorescence Open flowers Withered f lowcrs

No. infected No. heal thy

fruitlets fruitlets/others

No. infectcd No. heal thy

fru i t lets f ru i t lets/others

No. infected No. heal th l '

f ru i t lets f ru i t letskr thers

Leathery pocket

S 1

S4

Control

X2

-r4

1 3

l 0

850

930

I 004

90

93

76

9(l

I02

75

2 2 . 5 . . 1 . 2 N . S 2 . ( ) N . S

Cork spot

S I

S4

Control

XT

229

509275

655

+-1+

'739

20

5t3

l l

7 l

3n

Ot)

l - )

'7

7n5768

B lack eye

S I

S4

Control

x2

(.1

I

0

llu4

912

1 0 1 1

9 l

93

1'7

9 l

I 0 2

7 5

2 . 0 N . S 5 . 3 N . S 0 N . s .

Table 3. Pathogenici ty studies ofP. funiculosurn, strains S1, 52. 53 and 54

on PineaPple

TreatmenlMcan no . i n t ec ted cycs f r u i t

Leathery pocket Cork spot Black eye

57o) was used as an approximate guide to

determine whether the d i f ferences were

real ; the values for leathery pocket and

black eye were 4.7 and 0.3 respect ive ly .

Taking the difference of paired values for

leathery pocket severity in Toble 4 and

compar ing wi th the LSD (5olo) va lue of 4.7.

it was concluded that P. funiculosum Sl and

55, both of which were non-pigmented.were the causal agents of leathery pocket

d i sease . [ n t he same manner i t was c t l n -

cluded that P. funiculosum 56 (red-

p igmented, wi th shor t th ick c lub- l ikc

st rands) was the causal agent of b lack eye or

fruit let core rot Figure 2. Fu.sarium

moniliforme could also induce slight black

eye symptoms but was s igni f icant ly less

pathogenic than P. funiculosum 56 (Table

4) .

CONCLUSION

In Malaysia, the p ineapPle f ru i t

b lemish complex involv ing three commondiseases, viz. lealhery pocket. cork spot andfruit let core rot (or black eye) had been

S 1

S2

S3

S4

Control

4 .3 0 .03

r . 8 0 . 4 5

7 . 1 0 . 1 5

1 3 . 9 0 . l 3

3 .2 0 .0u

7 .33.u3 .34. t t1 . 4

LSD (54lc)

S .E . (mean )

3 . 5 N . S

1 . 1N . S

56 were compared with the earlier strains

isolated and F. monil iforme. The latter was

frequentf y isolated with P. funiculosumfrom fruit blemishes (Ltrul, 1983). The

resul ts showed that 55. which is non-pigmented, could a lso induce h igh levels ofleathery pocket disease although less

severely than 51 (Table 4). As the above

treatment plots were not replicated, thevalue of the 'variance of mean difference' xtr.,,. from experiment 2 (equivalent to LSD

169

Table 4. Pathogenicity of 5 strains of P. funiculosum incomparison with F. moniliforme on pineapple

Mean no. infected eyes/fruit

Treatment Leathery pocket Black eye

Inoc. before Inoc. duringanthesis anthesis

Inoc. before Inoc. duringanthesis anthesis

P. funiculosumS I

S2

S3

S5

S6

F. moniliforme

Control

1 6 . 9

7 . 6

- t - |

n . 21 . 4

1 . 9

2 .2

0.71 . 50.70.61 . 6

0.5

1 . 4

00004.0

0 .5

0

00003 .4

0 .2

0LSD (57,)(from Expt. 2)

4 .7

*Mean of 20 fruits/treatmen(

Figure 2. Fruitlet core rot symptoms (blackeye) after inoculation with BE30 (56).

shown to be caused by different morpho-strains of P. funiculosum. Leathery pocketis incited by the non-pigmented (S1 and 55)while cork spot is caused by the coremiaproducing 54. This is the first t ime that thecork spot disease is attributed to a distinctstrain of P. funiculosum.Fruillet core rot, iscaused primarily by 56, characterized by itscolony colour (pink and yellow) and theproduction of short club-like mycelialstructures, and to a lesser extent by F.monil iforme. In Hawaii, where anothercultivar, the Smooth Cayenne is grown,

both leathery pocket and fruit let core rotare caused by the same strain of non-pigmented P. funiculosum although F.moniliforme too could incite fruitlet core rot(RoHnencH and Pnprnne,R, 1976).

The presence of relatively low levels ofleathery pocket disease in control plots andin plots inoculated with the non-pathogenicstrains e.g. 52 and 53, was attributed to theubiquitous presence of the pathogenic Sland 55 in pineapple fields. Both the patho-genic strains were isolated from decayingpineapple trash, peat soil and water thatcollected in the centre of the plant (AuoN.,1981 ) .

The observation on the different.strains of P. funiculosum and F.monilifurme, their different modes of infec-tion and the different symptoms caused, hasfurther contributed to the existing know-ledge on the fruit blemish complex ofp ineapple.

The relatively low levels of leatherypocket disease obtained in some of theexperiments despite inoculation with thepathogenic isolates indicated the possibleinvolvement of one or more disposing

170

factors in disease expression. Work on this

aspect is in progress and the results will be

presented in another PaPer.

ACKNOWLEDGEMENTS

The author is grateful to Dr. Tay Tian

Hock, formerly Head of Horticulture

REFERENCES

ANoN. ( 1981 ) . Ann . Rep . f o r 1981 , F ru i t B ranch '

MARDI .

L rM . W.H . ( 1981 ) . Va r i e ta l eva lua t i on o f e i gh t

pineapple cul t ivars to leathery pocket d isease'

Tech. Pap. No. 24. Jalan Kebun: MARDI

(mimeo.) .

(1982). Leathery pocket d isease of p ineapple

and its control. Fruit Br. Info. Leaflet No' 13'7

pp. Jalan Kebun: MARDI (mimeo.) '

- (1983). Penicillium funiculosum isolates

Accepted for publication on 27th Februari, 1985r7l

ABSTRACT

Six strains of penicillium funiculosum and one of Fusarium moniliforme isolated from pineapple

fruit blemishes were evaluated ior their pathogenicity on pineapple fruit (cv. Masmerah) and for the

nature of the symptoms expressed. Different types of fruit blemishes were found to be caused by

different strains. Leathery pocket was causcd by two strains of P. funiculosurn with non-pigmented

reverse (S1 and 55). Fruitlei core rot (or black eye) was caused by a strain with pigmented reverse (56)'

while coik spot was caused by a reverse pigmented, coremia producing strain,54. Fusarium moniliforme

could only induce very l imited fruit let core rot symptoms. Two other pigmented strains' 52 and 53' wcro

non-pathogenic. The different strains were found to differ in their sites of infection.

Branch for his support and interest in theproject and to Mr. Chan Thiam Hwo' Mr.Khalid Mat Saad and Mr. KamilYaacob fortheir invaluable assistance in the field andlaboratory.

associated with fruit blemishes of pincapplc (cv'

Masmerah) . MARDI Res. Bu l l . l l ,179- t l6 '

L rv . T .K . and Rot tnse< ' r r . K .G. ( l9 t t0 ) . Ro lc o f

Penicillium funiculosum strains in thc dcvelop-

ment of pineapple fruit discase. Phynpathoktl4y70.663-5 .

RoHasrcu , K .G. and Pnr . t rn r .n . J B (1976) . F ic ld

induction of pineapple fruit let corking' lcathcry

pocket and fruitlet core rot with Penicillium

funiculosum. Phytopathoktgy 66, 392- 5'

Diterbitksn oleh:Instit iut Peny€li6ik8n d8n Kemajusn Pertanian Mslsysia {MARDI),

Peti Surat 202, U.P.M., Serdang, Selsngor, Malsysia.

Dicstsk oleh Percstakan Nadzam, Kuals Lumpur.