Pythium species are agroup of importantpathogens of floralcrops. Pythium dis-eases are typically

soilborne and are difficult to con-trol once a plant is infected orsoil/soilless mixes are contami-nated. These diseases usuallyattack root systems and progressgradually without apparent foliarsymptoms until a plant is nearcollapse. As a result, they oftenreceive less attention, are not like-ly controlled in a timely mannerand may result in more crop loss-es than those diseases displayingmore obvious symptoms. The beststrategy for managing Pythiumdiseases is prevention by elimi-nating or minimizing inoculumfrom all possible sources.

Among numerous sources ofinoculum, irrigation water can bean important one for Pythiumspecies, as already demonstrated

for Phytophthora species. Speciesof Pythium and Phytophthora areclosely related to each other mor-phologically and genetically; theyare in the same family taxonomi-cally. Pythium species are 10-100times more populous thanPhytophthora species in irrigationwater. But unlike Phytophthorapathogens, Pythium species in irri-gation water are poorly under-stood. This is partly due to theextreme difficulty in species identi-fication of Pythium. Basic informa-tion is lacking. For example, whatand how many species of Pythiumare present in irrigation systems?Are these species plant pathogenic?

Chlorination is an integral partof crop production in many enter-prises. A very practical andimportant question is whethercurrent chlorination protocols rec-ommended for Phytophthoramanagement can control Pythiumpathogens in irrigation water.

IN THE GREENHOUSECapturing surface water for

irrigation is critical to sustain theproduction and growth of indi-vidual enterprises and the floralindustry as a whole, as globalwater scarcity and pollutionrapidly spreads. How to mini-mize disease pressure associatedwith recycling irrigation water isan emerging and serious ques-tion all growers face today. Animmediate benefit of this studyis provision of a procedure toensure efficacy of chlorinationfor controlling Pythiumpathogens in irrigation water.This study clearly demonstratesthe diversity of Pythium speciespresent in irrigation water andthe need for water decontamina-tion in greenhouse and nurseryproduction, especially thoserecycling irrigation water.

You can use these results andstart developing lines of defense

3 2 GPN M a y 2 0 0 4

Pythium

pests & diseases

By Ping Kong,Patricia A.

Richardson and Chuan Hong

Reclaiming water may be cost efficient, but are you

doing the right things to prevent a Pythium outbreak?

RecycledIrrigation Water

and

along with chlorination againstpathogen build-up in recyclingirrigation systems through modi-fications or new designs. You canalso use these results to prioritizeproduction expansion andresearch agenda. This studydeveloped a molecular finger-printing technique for rapid andaccurate species identification.This will greatly facilitate futureresearch on biology and manage-ment of Pythium species anddiagnosis of plant diseasescaused by these species, and willhelp you improve disease man-agement in the long run.

RESEARCH RESULTSGenetic codes of biological

agents are stored in chemicalscalled deoxyribonucleic acid(DNA) or ribonucleic acid(RNA). For Pythium pathogens,these genetic codes are stored indouble stranded DNA. In thisproject, we developed a DNA fin-gerprinting technique for rapidand accurate species identifica-

tion. The underlying mechanismof this technique is to separatethe two complementary DNAstrands, then let individualstrands form secondary struc-tures (conformations). Underprescribed fingerprinting condi-tions, formation of secondarystructures depends on the DNAsequence thus can be a reliablecharacter for species identifica-tion. The fingerprinting tech-nique is called single-strandedconformational polymorphism(SSCP) analysis. With a singleDNA fingerprint, we were able todifferentiate 36 Pythium speciesassessed so far.

With this DNA fingerprintingtechnique, we have identifiedmore than 20 species of Pythiumfrom irrigation water. The mostabundant species include P. disso-tocum, P. torulosum, P. sulcatum andP. porphyrae. All identified speciesare plant pathogenic but vary indegree of aggressiveness. Theseresults indicate the diversity ofPythium species present in irriga-

tion water and the need for waterdecontamination in greenhouseand nursery production, especial-ly those recycling irrigation water.

A total of 15 isolates were test-ed with zoospores for chlorinesensitivity and were assigned anumber/letter combination.These isolates included six fromnonchlorinated irrigation water,five from chlorinated water andfour from diseased plants.Chlorine assay of each isolate wasrepeated twice. Efficacy of chlo-rine treatment was measured by

the average number of coloniesper petri dish with the mostcolonies per dish for control (0ppm). Numbers of colonies variedamong the repeated tests of thesame isolates. They also differedwith isolates from the samesources (nonchlorinated water,chlorinated water or plants).Differences among the isolateswithin the three sources remainunclear. Figures 1 and 2, below,illustrate the data; detail assaydata of the 13 other isolates wereomitted for simplicity. ç

M a y 2 0 0 4 GPN 3 3

0

5

10

15

20

25

30

35

43210

No

.C

olo

nie

s

Chlorine (ppm)

Figure 1. Chlorine sensitivity of Pythium aphanidermatum (#5J5) isolated from a

Figure 2. Chlorine sensitivity of Pythium sulcatum (#15J9) isolated from irrigation water.Each line represents results of three repeated tests.

0

5

10

15

20

25

30

35

40

43210

No

.C

olo

nie

s

Chlorine (ppm)

Recycling water may put you at risk of a Pythium outbreak, but there are steps you can taketo prevent it.

Need a new sprayer for spring? DRAMM has all of the bases cov-ered:• Autofog• Coldfogger• PulsFOG• NEW Hydra Line of Hydraulic

pests & diseases

INTEGRATED PEST MANAGE-

sponsored by:

800.258.0848information@dramm.comdramm.com

will effectively control Pythiumzoospores in irrigation water.

It must be noted that substan-tial numbers of colonies wererecovered at 1 ppm for a majori-ty of the isolates, and there

Write in 712

No colonies were recovered at 2ppm or above for the majority ofisolates. Four isolates produced afew colonies at 2 ppm or above inone or two of the three repeatedtests. Isolate 17C2 produced twocolonies at 2 ppm in one test.

Isolate 23J3 produced 0.3 coloniesat the same concentration in twotests. Isolates 4E1 and 5J5 produceda few colonies at 8 ppm in one test,but none at 2 ppm or above in theother two tests (see Figure 1, page33). Isolates producing colonies at 2

ppm or above produced coloniesonly at 1 ppm or lower in addition-al tests. These results suggest thatthe previously recommended 2ppm free chlorine at dischargepoints (risers or sprinklers) for con-trol of Phytophthora species also

3 4 GPN M a y 2 0 0 4

pests & diseases

FloricultureI n d u s t r yR e s e a r c hand Scholarship Trust (FIRST), acombination of Bedding PlantsFoundation and Ohio FloricultureFoundation, supports floriculturescholarships and research, suchas this article, across the UnitedStates and Canada. Moreresearch results, scholarshipwinners and applications forscholarships or research fundingare available at www.firstinflori-culture.org. How can you help inFIRST’s drive to improve the pro-duction and marketability ofplants? Here are some ideas:

• Review scholarship andresearch grant applications• Purchase Gator GiveawayRaffle Tickets • Participate in the Dramm 5kRelay or DS Cole Hockey inColumbus events at the annualOFA Short Course• Become a board member• Participate in our fun tradeshow booths!!• Set up a named scholarshipor research fund• Purchase Reverse Raffle tick-ets for a chance to win $10,000and other cash prizes• Catch some laughs at thePaul Ecke Jr. Floral DesignContest during the annual OFAShort Course

For more information, you canalso contact FIRST, P.O. Box 280,East Lansing, MI 48826-0280;phone (517) 333-4617; fax (517)333-4494 or E-mail: first@firstinfloriculture.com.

essentially were no differences incolony recovery between 0.25and 1.0 ppm of chlorine for mostisolates. This chlorine response isconsiderably different from thatof Phytophthora species reportedpreviously. The mechanismbehind this is unknown. Butthese results suggest that anyfailure to maintain recommend-ed 2 ppm chlorine may exposeentire crops in the productionfacility to Pythium diseases.

Pythium diseases are a majorlimiting factor affecting floralcrop production. This problemwill continue to aggravate withgrowing global water scarcityand pollution. Identificationalong with pathogenenicity testsof Pythium species present inirrigation water suggested thatdecontamination of recycledwater before use is required toproduce quality plants. Growersshould use this recommendationas a guide when building a newproduction facility (e.g., green-house) or modifying an existingfacility to minimize Pythiumdisease risk.

IDENTIFICATIONIdentification of these species is

the first and most important steptowards development of effectivepathogen management strategies.This research also made specific rec-ommendations on using currentchlorination protocol for water-borne Pythium control. Use of theserecommendations will reduce therisk of incomplete water decontami-nation, which subsequently reducescrop losses and improves plantquality. In addition, this researchovercame a major difficulty in iden-tification of Pythium isolates intospecies, and produced a moleculartechnique that with a single DNAfingerprint steadily separateddozens of species. Accurate speciesidentification is foundational forany biological study. Lack of effec-tive identification tools has longprevented Pythium disease controlresearch from progressing as need-ed. This research providesresearchers with an effective tool fordisease management studies,improving research quality and pro-ductivity and helping the industryon Pythium management in thelong run. GPN

Chuan Hong is an assistant profes-sor of Plant Pathology, Ping Kong isresearch scientist and Patricia A.Richardson is research specialist atVirginia Tech’s Hampton RoadsCenter, Virginia Beach, Va.

This article was reprinted with per-mission from Floriculture IndustryResearch and Scholarship Trust.

M a y 2 0 0 4 GPN 3 5

pests & diseases

LearnMoreFor more informationrelated to this article, go to www.gpnmag.com/lm.cfm/gp050404