Indian Phytopath. 53 (I) : 50-56 (2000)

Cedrus deodara root rot disease-threat to the Himalayanforestry and environment

LAL SINGH and T. N. LAKHANPAL'Himalayan Research Group, Umesh Bahvan, Chotta Shim/a, Shimla 171 002

ABSTRACT: Studies on root rot disease of deodar (Cedrus deodara) caused by pathogen Phytophthora cinnamomi arebeing reported for the first time from Himalayan region. Disease resulted in wide spread mortality of deodar trees nearChail, Shimla. About 200 trees have completely dried up, and another 150-200 are in different stages of withering.Whereas the grown up trees face this disease problem, the young regenerating plants are healthy in spite of ampleinoculum in the soil. Extensive ectomycorrhizal presence in such plants acts as the main deterrent to invasion bypathogen. In older trees, where ectomycorrhizal mantle is lost from short roots, the surface gets exposed to attack byinvading pathogen. The pathogen produces zoosporangia in the soil extract each releasing 30-40 kidney shaped zoospores.This is the first report of any fungus causing such a large-scale disease of forest trees in Indian Himalayas.

Key words: Cedrus deodara, root rot, Phytophthora

Cedrus deodara, the state tree of Himachal Pradeshis one of the most important conifers in the Himalayanmoist temperate forests. It extends from Afghanistanthrough the hilly regions of Pakistan and India toKurnauli valley in Nepal occupying an elevation of1200-3050 m. The total area of deodar forests in Indiais estimated to be around 203,263 hectares. Out ofthis, 69,872 hectares lie in Himachal Pradesh (Anon,1976). Deodar forests play an important role in theeconomy of the country, as its wood is the strongestand most durable. Further, the elegant trees of C.deodara add to the scenic beauty of the Himalayas.Disease aspects of this valuable tree till date are sub-jected to only scant studies. A search into literaturereveals that on Iy a few diseases alongwith causal or-ganisnis were listed and described by Maheshwari andBiswas (1970) and Bakshi (1976). Hence, it becomesdifficult to decide if any unreported disease conditionis observed and whether it is caused by a native or anintroduced pathogen. Introduced diseases, when allowedto run unchecked, can affect the aesthetic and thus therecreational value of the forest, which is of prime im-portance in the Himalayan region, in general, andHimachal Pradesh in particular. Schematic presenta-tion of the infected forest is given in Fig.l.

This paper, for the first time, reports studies onroot rot of C. deodara caused by Phytophthora

IDepartment of B io-Sciences, H. P. Uni versity,Shimla 171 005

cinnamomi in Chail forest of Himachal Pradesh wherevast stretch of deodar trees was observed to undergounnatural drying. The fungus caused serious damage,devastating around 7-8 ha of pure deodar forest pos-ing challenge to the economy, ecology and environ-ment of the area.

MATERIALS AND METHODS

Site description

The affected deodar forest is located about 2-2.5km, from famous tourist destination Chail in the out-skirts of Shimla City at about 1950 metres above MSL.Deodar is the dominant species in the entire forest andforms almost pure stands. The affected forest on the.first look appeared as if it has been destroyed by fire(Fig 2a). Almost all the mature trees in the area ex-tending to about 5-7 hectares were dried, and died andothers are standing in various stages of decay. Whereas,some of the dead and dry standing trees still have thebranches and bark intact, the advanced stages of decayare marked by gradual shedding of branches and bark.In some of the decayed trees, the exposed wood be-neath the bark bears lesions indicating that trees musthave been dead for quite some years. Local peoplecorroborate these observations stating that the decayprocess started about 18-20 years back.

Sample collection

In the infected forest trees were found in differentstages of disease development. The infected trees were

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• Compldtly dry trtt.s in :ldnaced it_lei of dec.y.

t TrttS wilh final J"b.~ Jymptoms witb cblorotk and IOOt1 Jtaws.

ilIullhy trees.

Fig.l. Schematic presentation Cedrus deodara forest in-fected with Phytophthora cinnamoni

grouped into eight categories: (i) debarked in advancedstages of decay, (ii) dried and dead trees with somebark intact and still to undergo decay, (iii) dried anddead trees with bark and branches intact but withoutneedles, (iv) dried and dead 'trees with bark, branchesand dry needles intact, (v) dried and dead trees withbark, branches and needles intact but defoliation ofneedles has just started at the ends of branches, (vi)living trees with chlorotic foliage and no defoliationyet. (vii) trees without any visible symptoms of diseaseand (viii) young regenerating healthy plants. Soil, rootand wood samples of all eight categories were col-lected in autoclaved polypropylene bags in triplicate.

Fungal isolation

For isolation from diseased roots, wood, bark andsoil, potato dextrose agar (PDA) medium was used.The infected root pieces were washed briefly in water,dipped for a few seconds in 70% alcohol, blotted on apre-sterilized Whatman filter paper and placed en PDAin petriplates and slants in test tubes. Fo~ isolationfrom wood and bark, a shallow cut was given on thesurface of wood and bark. and then these were brokeninto pieces by applying pressure with fingers along thecut exposing the unexposed uncontaminated portion.From this freshly exposed portion, a piece of tissuewas quickly lifted with forceps and placed on themedium in petriplate. Culture isolation was done di-rectly from the soil crumbs and by soil dilution methodon PDA having streptomycin sulphate to inhibit soilbacteria and fungi. Soil crumbs were directly sprinkledon the surface of petriplates containing PDA. Soil di-lutions were prepared by mixing 19 of soil in 100 mlof sterilized distilled water. For isolation on petriplates,

Indian Phytopathology 51

1ml of the solution from 1:4 dilution was used.Petriplates were incubated at 23°C for 72 hours underdark conditions.

Isolation of chlamydospore

Chlamydospores are released into the soil follow-ing decay of roots. Chlamydospores were recoveredand recorded from soil by dilution plate method anddirect plating of soil crumbs. Dilutions were preparedfrom the suspension of one g soil in 100 ml of steril-ized distilled water.

Zoosporangial production

Zoosporangial production was tried in non-sterilesoil leachate as described by Mehrlich (1935) with slightmodifications. Colonies of cultures were produced on100 ml potato dextrose broth (PDA-agar) from 8mmculture discs. After four days of growth, colonies wererepeatedly washed in sterilized distilled water to re-move the unutilized nutrients. Colonies were trans-ferred to flask containing 100 ml unsterilized soilleachate prepared by mixing 10 g soil in 100 ml dis-tilled water and filtering through Whatrnan No.4 I filterpaper. Flasks were incubated at 23°C

Morpho-anatomical details of root

Roots were washed in running tap water to re-move the adhering soil particles. Roots were observedfor morphological details under stereo microscope. Foranatomical studies, roots were fixed in formalin aceticacid and 70 % alcohol (5:5:90) for 24 hours and storedin 70 % alcohol. Sectioning was done with hand usingrazor blade.

Pathogenicity test

Pathogenicity of the isolates was tested throughKoch postulates. Healthy seedlings of Cedrus deodarawere inoculated with the pathogen under glass houseconditions. Mass inoculum of pathogen was prepared'in two litre flasks containing one litre of potato dex-trose broth. Flasks were inoculated with 8 mm discs ofculture. Pieces of broken glass were placed in broth forthe maceration of mycelium through constant stirringfor 1-2 minutes every day. Flasks were incubated forone month in incubator at 25 ± 1°C. After one monthmedium was decanted off and macerated mycelial cul-ture was thoroughly washed with sterilized distilledwater 2-3 times to remove any unutilised nutrients.Mycelium harvested from three flasks was mixed intwo litres of sterilized distilled water. C deodara seed-lings were uprooted from the polythene bags and di-rectly dipped in this mycelium slurry, slightly smeared

52 Indian Phytopathology

SO that mycelium adheres to the roots. These werereplanted in the polythene bags. Uprooted seedlingsdipped in plain sterilised distilled water served as con-trol.

RESULTS

Symptoms

The disease has resulted in the decline and deathof trees above IS" diameter at breast height (DBH) andolder trees. The foliage in the infected trees turnsyellowish, the length of needles and shoot is reducedand finally the diseased trees become conspicuous withscant and chlorotic foliage extending in centripetalmanner (top to base and from end of branches towardsthe stem). Cone size is also considerably reduced. Thetops of the trees and tips of the branches dry up (Fig2b). The photograph shows localised disease in a patchof deodar trees. Below the diseased patch runs theforest road and below this road all the trees are healthy.The road, therefore, seems to have checked the spreadof the disease. In the backdrop of infected patch, natu-rally regenerating plants of C. deodara are clearly vis-ible. All these are healthy plants. On investigation, allthese ·were found to have well-developedectomycorrhizal roots, which apparently deter infec-tion by the pathogen:

Fungal isolation

The culture of P. cinnamomi isolated from the soiland infected roots of C. deodara is white in colour andthe colonies exhibit characteristic rosette growth pat-tern (Fig. 2d). Fungus could be easily isolated fromsoil and roots from all samples except roots of com-pletely dried trees showing advanced stages of decayand from roots of young regenerating plants .. Even thesoil from near the vicinity of the trees without visiblesymptoms had the same quantum of inoculum as thatnear the highly diseased trees. No inoculum was,however, present in the wood and bark of any categoryof trees in the diseased forest. Detail of fungal isola-tion from different parts of the' trees, showing symp-toms of disease development in different stages, is givenin Tablel.

On microscopic examination, fungus has beenfound to possess two types of well-developed profuselybranched, hyaline mycelia, which are coenocytic andmoderately thick. Subterranean mycelium is composedof hyphae 5-6 urn in diameter. Cytoplasm contains scat- .tered nuclei. Older hyphae occasionally undergo sep-tation. The fungal hyphae ramify the intercellular spacesof the cells of host root tissue. Ageing cultures produce

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Table 1. Isolation of P. cinnamomi from different parts oftrees showing symptoms in different stages ofdevelopment

Sample Category Soil Root Bark Wood

Debarked trees in advanced +stages of decay

Dried and dead undecayed + +trees with some bark intact

Dried and dead trees with + +bark and branches intact butwithout needles

Dried and dead trees with + +bark, branches and dryneedles intact

Dried and dead trees with + +bark, branches and needlesintact but defoliation ofneedles has just startedat the end of branches

Living trees with chlorotic + +foliage and no defoliation yet

Trees in the diseased patch + +without any visible symptomsof disease

Young regenerating healthy +plants with mycorrhiza

+ P.cinnamomi present; - P.cinnamomi absent.

a number of chlamydospores on lateral hyphal branches.Chlamydospores are thin walled, globose 20-45 urn indiameter. Chlamydospores germinate through multiplegerm tubes (5-7) on potato dextrose agar medium. Nosex organs have been observed in culture, however.

Chlamydospore isolations

Isolations from soil made through dilution platetechnique were directly observed under stereo micro-scope. Germinating chlamydospores were frequentlyobserved in the samples. The chlamydospores in thepetri plates were observed to be without any germina-tion activity. The number of germinating chlarny-dospores was more in soil collected from recently driedtrees and trees showing initial stages of disease devel-opment. The soil samples collected from the dry treeswith advanced stages of decay exhibited comparativelyless germination activity. of the chlamydospores. No

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isolationof chlamydospores was achieved from the barkand wood samples of any category of trees.

Sporangial production

Sporangial production is more complicated in P.cinnamomi than other species of this genus. Sporangiaof P. cinnamomi are non-papillate. They are generallyellipsoid to ovoid in shape, 20.0-35.5 urn wide x 55.0-

Indian Phytopathology 53

Fig. 2. (a) Dead and dry trees ofC.deodara in different stagesof infection; (b) Patch of in-fected trees of C.deodara inthe forest with chlorotic foli-age; (c) Cedrus deodara rootsshowing fungus mycelium anddead short roots; (d) Isolatedculture of Phytophthoracinnamomi showing character-istic rosette growth on PDA;(c) Healthy roots ofregenerat-ing seedlings with conspicuousectornycorrhizal development;(f) T. S. of ectomycorrhizalroot showing thick outer fun-gal sheath (mantle); (g) T. S.of uninfected short roots., (h)T. S. of diseased short root.

75 urn long. Each sporangium produces 30-40 kidneyshaped biflagellate zoospores, each 10- 12 urn in diam-eter.

I

Morpho-anatomical details of rootsPlate I e shows the morphology of mycorrhizal

roots in a healthy regenerating plant of deodar. Theectomycorrhiza covers the roots entirely, is cream ish

54 Indian Phytopathology

white in colour and is branched in a corralloid manner.In T. S., such roots reveal the presence of a 45-60 11mthick mantle (Fig. 2f) and well developed Hartig net(Fig. 2f). Fig. 2g is the T. S. of a short root, from anolder infected tree; the root is yet to be infected andT. S. reveals the normal root anatomy but there is nohyphal mantle nor there is any Hartig net. Plate I h isthe T. S. of an infected short root showing its fate afterinfection with the pathogen. The root is shrivelled, allthe component tissues are deformed, there is lot oftannin deposition in the cortex and the vascular tissueshows lignification. Ultimately, the roots acquirecharcoal black colour. Fig. 2c shows the profuse growthof the fungus under bark of the mother roots. Theshort roots are also infected. The fungus frommother roots and directly from the soil and adjacentinfected roots extends to the lateral short roots andcolonises the inter and intra-cellular spaces includingvascular tissues.

Pathogenicity test

Pathogenicity test was performed following stepsproposed by Koch (1876). P. cinnamomi culture wasisolated from the roots of infected C.deodara trees.Pure culture was multiplied in PDA broth in flasks. Itwas compared with the original culture for purity. Seed-lings inoculated with mass culture developed chloroticleaves, shorter in length and which dried completelywithin six months of inoculation. Symptoms appearedat the onset of spring season. There was reduction inleaf size, chlorosis of leaves and absence of new shootsin the seedlings inoculated with pure culture of fungusas observed in the infected forest trees. Short roots inartificial inoculated seedlings were dark brown to blackin colour resembling short root collected from infectedforest trees. A comparison of reisolate with originalisolate reveals them to have same characteristics con-forming similarity of the two and proving the pathoge-nicity of. the fungus.

DISCUSSION

The species of Phytophthora are well known patho-gens of a wide range of hosts the world over. In India,so far Phytophthora cinnamomi had been reported onlyfrom Andhra Pradesh causing root rot of grapes(Agnihothrudu, 1968) and from stem and roots ofCincona from Anamalai, Tamil Nadu (Ramakrishnan,1951). Other species of the genus Phytophthora havebeen reported to cause diseases of agricultural andhorticultural crops. Infection of conifers in theHimalayas by any species of Phytophthora, is first everreport on deodar. P. cinnamomi was first described byRands (1922). Now it is known from over 60 coun-

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tries causing, infection of more than 900 host plants(Zentmyer, 1980). The same fungus had earlier earnednotoriety for wiping out Jarrah tree (Eucalyptusmarginata) in western Australia causing "jarrah die-back', It also causes short leaf disease of short leafpine (Pinus echineta) in the United States. The fungusis now having repeat performance of notoriety earningthe same bad name in mild temperate, subtropical andtropical areas by infecting woody plants.

In deodar, the infected deodar plants developedchlorotic foliage with considerable reduction in leafsize. This is essentially a reflection of nutrient defi-ciency which, results due to the death of short absorb-ing roots and poor regenerative capacity of the ageingmain roots. Growth of P. cinnamomi was noticed to be .more vigorous during rainy season when there is plentyof moisture and the temperature is optimum for thegermination and movement of fungal propagules, whichare motile zoospores. During infection, most of theshort roots are infected and rendered dead, by the timenext spring season sets in. Demand of plant for higheruptake of nutrients in the season is not fulfilled, whichultimately results in the abrupt appearance of chloroticfoliage and decline in the growth of trees. In seedlingstage, death may occur within a few days of the ap-pearance of symptoms but in mature trees, 15-20 yearscan lapse before they actually succumb. Pathogen cansurvive, and multiply saprophytically on dead organicmatter. The disease can also spread from one tree toanother by root contact or by zoospores in the presenceof water,

In the area under study, the young regeneratingplants of deodar were healthy and their roots were alsonot infected even though the soil where these weregrowing had enough inoculum of P. cinnamomi. Ex-amination of such roots revealed that these roots wereprofusely ectomycorrhizal (Fig.2e) with thick hyphalmantle (Fig. 2f). This suggests that the ectomycorrhizalmantle was acting as a mechanical barrier, not allow-ing the entry of pathogen. The older trees were how-ever, seriously infected. In older trees, short roots werewithout ectomycorrhizal sheath (Plate 1 g) and hencethey were more prone to infection by the pathogen. Ithas also been reported that breakdown of mycorrhizalprotection may also occur during periods of prolongedrainfall (Bassett and Will, 1964; Marks, 1965; Newhook,1959) by bacterial lysis (Foster and Marks, 1967) andby the rupture of mycorrhizal mantle during root elon-gation ( Marks and Foster, 1967)' making the rootsvulnerable to infection.

In some cases, mycorrhizal fungi and microorgan-isms harbouring the soil around short roots are reported

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to produce some antifungal and antibacterial com-pounds, which inhibit root pathogenic infections (Marx,1971). However, the significance of antibiotic produc-tion by saprophytes in reducing the inoculum potentialof root pathogens and of subsequent root disease de-velopment is not yet fully understood.

Detailed studies related to specific interaction be-tween the mycorrhizal fungi and P.cinnamomi onloblolly pine have been published by Marx and Davey(1969a, 1969b). These studies contain substantial evi-dence showing the role of ectomycorrhizae as biologi-cal deterrent to the infection of roots by pathogens,including P. cinnamomi. Complete absence of anyapparent disease symptoms from the young regenerat-ing deodar plants in the affected forest area and thepresence of vigorously growing ectomycorrhizae ontheir roots are clear indications to the ectomycorrhizalassociation acting as physical barrier to the infection ofthese plants by the pathogen.

Another interesting feature of this study is that P.cinnamomi propagules (mainly chlamydospores) weredetected in advance of symptom development. P.cinnamomi was isolated from the asymptomatic treessampled in the forest adjacent to infected trees whereno mortality had yet occurred. The regular cattle graz-ing and human trespassing observed during samplingsin the infected forest may be one of the probable meansof inoculum dissemination to the adjoining areas. Thisobservation necessitates the need to collect soil samplesbeyond the areas of mortality to determine the extentof spread of the pathogen to optimize disease controlprocedures.

The wide host range of the pathogen and faculta-tive mode of parasitism ensure the survival of this patho-gen in one form or another, thus making control mea-sures difficult. Localized fumigation of soil has beenused to check the disease but this cannot be/adoptedfor vast stretches of forest land. Pratt (1971) suggestedthe possibility of controlling P. cinnamomi by naturalor induced antibiosis. Newhook and Podger (1972) havediscussed in detail this approach for control on a largescale, but still a lot more research is needed to achievethe desired results. Marx (1973) concluded that in allprobability most of the mechanisms of root protectionof mycorrhizae are functional at any given time sinceseveral appears to be inseparable (i.e, mantle barriers,host origin inhibitors, differences in chemical exuda-tion, etc.). This broad spectrum of defence mechanismsacting in concert assumes greater opportunities for bio-logical control of root pathogen by mycorrhizae.

Keeping in view the reported severity of P.

Indian Phytopathology ss

cinnamomi infection from other countries, it is impor-tant to take all precautions against the further spread ofthis deadly disease in the north-western Himalayas.

ACKNOWLEDGEMENTSThe authors gratefully acknowledge the help ren-

dered by Prof. K,G. Mukerji, Department of Botany,University of Delhi for authentication of the fungalculture.

REFERENCES

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Bakshi, B.K (1976). Forest Pathology: Principles and Prac-tices in Forestry. Forest Research Institute PressDehradun. pp 400.

Bassett, C. and Will, G. M. (1964). Soil sterilisation trialsin two forest nurseries. New Zealand 1. Forest. 9 : 50-58.

Foster, R. C. and Marks, G. C. (1967). Observations onthe mycorrhizas of forest trees II, The rhizosphere ofPinus radiata O.Oon. Aust. 1. Biol. Sci. 20 : 915-926.

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Marx, D,H. and Davey, C. B. (1969a). Resistance of asep-tically formed mycorrhizae to infection by Phytophthoracinnamomi. Phytopathology, 59 : 549-558,

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Received for publication February 24, 1999