Proceeiinq« of the 7th International Worknlg Conference an Stored-product Protecuon - Volume- 2

Seed-bearing and seed-transmission of soybean phytophthoraroot rot

Zhou Zhaohui and Yan ] mi

Abstract

Soybean phytophthora root rot caused by PhytophthorasOJae Kaufmann & Gerdemann is a typical fungal diseasetransmitted by soil. It was unknown whether the pathogencould be transmitted by soybean seeds, and there were noreports about seeds test for tlus disease Seed transmission,the morphology and the location of the pathogen withm theseed and the techmque of seeds test used for plantquarantine ~ere descnbed m detail m this paper. It showedthat the plants infected by Phytophthra eoiae could produceinfected seeds. It also demonstrated that soybeanphytophthora root rot could be transmitted by infectedseeds.

Introduction

Soybean phytophthora root rot caused by Phytophthoraecnoe : Kaufmann and Gerdemann is a dangerous disease onsoybean, and leads to the decaying of seeds, damping off ofseedlmgs, and rotting of roots and stems rot This diseasegenerally can cause yield loss of 25 - 50% on susceptiblecultrvars, and senously 100% Soybean phytophthora rootrot is a typical soil-borne disease, but seed-bearing and seed-transnussion have not been concluded because there is fewreference abo~t__it, ~ that some SCientists take a suspectattitude The problems of seed-beanng and seed-transnussion should be solved urgently for plant quarantineThis paper presented a senes of studies on seed-beanng andseed-transnussion of soybean phytophthora root rot

Materials and Methods

Seed-bearing study

Funga l culturesPhytophthora sojae race 5 was obtained from Department

of Plant Pathology, Iowa State Urnversrty , USA; S-317 wasobtained from Department of Plant Pathology, Chma

1 Institute of Plant Quarantme, Mmistry of Agnculture, Hmxmh241, Chaoyang Thstnct, BeIJmgtlO0029, P R Chma

Agnculture University. HH-, and HM- were obtained fromHeilongjiang Province, China.Soybean seedlmgsYouBian30 was obtained from Department of Agronomy,

China Agnculture Unrversity: Clark, Harosoy, Harosoy 63were obtained from Institute of Crop Germplasm Resources,China Academy of Agncultural Science; Hefeng 25 andSumong 8 were obtained from Heilongjiang Institute ofAgncultural SciencesArtificzal inoculaiu»iArtificial inoculation was made With the followingmethods

after healthy seedlmgs were grown in stenhzed soil ingreenhouse1) Wound moculationIn the growth penod of pods-forming of soybean plants,

the surface layer of stems and branches was scratched Withscalpel, then the hyphae mass of 1x 4 rom, which has beencultured on CA for 7 days, was inserted into the woundedSite, and kept m high hunudity for 7 days, Withno pathogenwounded as control. Tlus test had several replications.2) Injection moculationThe husks were injected With O.2ml zoospore suspension

(about 5 x 102/ml), With stenhzed distilled water ascontrol Infection was observed 7 days later under the roomtemperature m high humidity ThIS test had severalrephcanons3) Filter paper disc moculationStenhzed- filter paper dISCS(4mm m diameter ) were

immersed m zoospore suspension (about 5 x 102/ml), andthen attached to the surface of the husk enveloped mabsorbent cotton, With stenle distilled water as control.Infection was observed 7 days later under the roomtemperature in high humidity. This test had severalreplications4) Soil moculationHealthy seeds that began to gemmate were grown m

inoculated soil under the room temperature, and werewatered to keep high humidity Infection was observed afteremergence This test had 2 replicationsExaminatum of mfected seeds1) Isolation of the fungi m infected seedsArtificial inoculated seeds were isolated on selective

mediUm (LA-PARPH), Limabean mediUm(LA) and m tapwater. One moculated seed was separated mto two portions,

1802

Proceedings of the Tth Internatumal Working Conference Oil Stored-product Protection - Youune 2

one was Isolated Immediately after bemg harvested, anotherwas Isolated 8 - 12 days under the room temperature afterharvested. This test had several replications2) Examination of mfected seedsInfected seeds were Immersed m 10% KOH for 4 hours,

the spermoderm, embryo and cotyledon were surveyed forthe pathogen m morphology under the rrucroscope ThIS testhad several replicationsPathogenicdy of nifecteii seedsInfected seeds were harvested from nature field plants,

every part of these seeds (embryo, cotyledon andspermoderm) and husks were Isolated The Isolated cultureswere surveyed for the pathogen m morphology and weredetermmed for pathogenicity accordmg to Koch's postulates.The seedmgs of Hefeng 25 and Sumong 8 were artrficiallymoculated with the pathogens from mfected seeds and 3cultures from mfected plants and race 5 from USA by usmgwound moculation in the greenhouse, WIth no pathogenmoculation as control. These tests had 2 - 3 replications,sometimes 4 - 5 replica tions

The test of seed-transmission

Infected seeds were planted directly1) Artificial moculated seeds were separated mto twc

portions: (A) 7 complete mfected seeds; (B) 10 mfectedseeds which were separated mto spermoderm and cotyledonA and B were put mto stenhzed soil WIth about 30 - 40%

Table 1. The result of artificial moculation.

water content m small beakers respectively, then removedto sterilized soil 48 days later under the room temperature,the seeds of Hefeng 25 that began to gemmate were grownm such SOlI The pots were Immersed m tap water for 36hours after all seeds emerged Infections were observed.2) Isolation of mfected plants from mfected seeds and Its

pathogerucity: Infected plants were Isolated on selectivemedium (CA-PARPH) and m tap water Pathogenicity wastested by usmg Isolated cultures

Results and Discussion

Artificial inoculation

Artifrcral mocula tion tests were made for 41 times dunng1992 - 1993 Infected rate of soil inoculation was 100%,and all the inoculations of wound, dISCand injection resultedin infections (showed m Table 1). 300 mfected pods wereharvested. No infection occurred m the controls The podsat the SIte of the mfected stem could be mfected m theexamination of wound inoculation. Water-soaked symptomspread from the bottom to the top on the pod, fmally thewhole pod became yellow WIth wnnkled dry and no lusterseeds ThIS expenment demonstrated that seeds could bemfected when the pod was mfected by Phytophrhora sojaeA number of such seeds could carry the pathogen when thestem was mfected

Artificial mocula tion infected

moculated method year&CK times No of plant No No. mcidence % mocula ted sites

1992 9 31 178 137 77 stem, branch

wounded CK 6 6 6 a a stem, branch

inoculation 1993 2 9 21 21 100 stem, branch

CK 2 2 2 a a Stem, branch

1992 17 79 303 288 95 Husk

mjection CK 12 12 12 a a Husk

1993 4 20 35 25 71 Husk

CK 4 4 4 a a Husk

1992 2 9 30 5 17 Husk

DISC CK 2 3 3 a a Husk

Inoculation 1993 5 16 44 21 48 Husk

CK 5 5 5 a a Husk

Soil 1992 2 19 19 19 100 Seed

Inocula tion CK 2 10 10 a a Seed

Note: No of plant: Youlnanl. 30; Clark: 70; Harosoy: 8; Harosoy: 25, Total:133

1803

Proceed~ngs of the 7th Internatumal Workmg Conference on Storei-product Protectwn - Volume 2

Examination of seed-borne

Isolation of seed-borne pathogenPhytophthora scnae was obtamed from mfected husk,

spermoderm, embryo and cotyledon after 40 mfected podsand 96 infected seeds were isolated. No Phytophthora sppwas obtained from 7 healthy pods and 12 seeds.Isolating Infected seeds showed that the pathogen could be

obtamed from embryo, cotyledon, spermoderm and husk Ifinfected seeds were isolated immediately after beingharvested, no pathogen was obtamed from seeds 8 -12 daysafter bemg harvested (see Table 2). These tests also drew aconclusion that It was very difficult to Isolate Phytophthrasojae from dry infected seeds. The results of Isolation onselective medium and in tap water were almost the same,

and selective medium was much better for pure culture.Examination. of seed-bearingThe exanunation of every part of the mfected seed

( spermoderm, embryo and cotyledon) showed that thepathogen survived as oospores, oogoma and hyphae mseeds, oospores m spermoderms only and hyphae m embryosand cotyledons. 35 mfected seeds that were artificiallymoculated 17 - 23 days after bemg harvested were examinedand 12694 oospores were found m spermoderms.The pods near the stems wounded could produce infected

seeds, the symptom could develop up and down under thefavorable condition which caused pods to be mfected andproduced Infected seeds These tests demonstrated that thepathogen could spread m the conductmg tissue of plant tomfect pods and seeds

Table 2. The result of Isolation of mfected seed.

Inoculated LA-PARPH (LA) Water

Cultivar Method sequence embryo cotyledon spermoderm husk embryo cotyledon spermoderm husk

1 +++ +++ +++ ++ ++ + +t:

YB30 DISC 2 +++ +++ / + /

2'

3 +++ +++ +++ +++ +3'

1 +++ +++ +++ ++ +++ +++ +++ +YB30 Wounded l'

Stem 2 +++ +++ +++ +++ +++ +++2'

1 +++ +++ +++ ++ + +++ +l'

Clark Injection 2 +++ ++ +++ + + +2'

3 + + +3'

+++ +++ +++ + + ++ +YB30 Wounded l'

Stem 2 +++ +++ +++ +++ +++ +++2'

Below 1 +++ +++ +++ + +++ +++ +++YB30 Wounded l'

Stem 2 + + ++ +++ ++2'

Note: (1)1,2,3 indicate that a half of the mfected seed was isolated immediately after harvested; I: ,2' ,3' indicate that another half of themfected seed was isolated 8 -10 days after being harvested@' + "mdicate Phytophthora sOJae has been isolated, '- "mdicate Phythophthora sojae has not been isolated

1804

Proceedtng8 of the 7th International Working Conference on Stored-product Protection - Volume 2

stem from one infected plant had the same pathogemc to thesusceptible vane ties (Table 3)

Experiment of seed- transmission

Pathogenicity of seed-borne pathogen

The fact that the infected seeds produced at the infectedstem dunng the growing penod of pods forming under thenatural condition was found dunng the field mvestigation mAugust of 1993. There were 22 infected plants with 56mfected pods from 96 infected plants, which was WIth anaverage of 2.5 infected pods. After the isolation of infectedseeds and husks mentioned above, 18 cultures ofPhytophthora 80jae were obtained, from which, 12 typicalcultures and 3 cultures from infected stems were selected totest pathogenicity. The result showed that the pathogenIsolated from any parts of the infected seed hadpathogemcity and the pathogen Isolated from seeds(mcludmg spermoderm, cotyledon and embryo), husks and

SIX infected plants were obtained m two expenments ofseed- transnussion, one of them came from the infected seedItself; another arose from infected spermoderm, the other 4infected plants arose from seed-transmission (A), themfected rate of seed-transrmssion was 5 - 21 % (Table 4).SIX cultures of phytophthora 80Jae were Isolated fromstems, basal stems and cotyledons etc. of the infected plantfrom seed- transmission. These cultures were kept in thePhytophthora Laboratory, Institute of Plant Quarantme.Both expenments demonstrated that the pathogen could betransmi tted by seeds

Table 3. The result of the pathogemc for seed-borne pathogen.

pathogen No. of No. ofincidence

cultrvar Inoculated infected%

indexcode source Isolated site plant plant

HH-819-3 stem HF25 17 17 100 100

Aa- SN8 20 20 100 100HH-819-3 cheng cotyledon HF25 5 5 100 100A-2 SN8 5 5 100 100

HF25 20 20 100 100HH-820-1 cotyledon SN8 20 5 25.0 21.7B-1 Suburbs

HH-820-1 of Harbin husk HF25 20 20 100 100C SN8 19 4 21.1 12.3

HH-821-2' spermoderm HF25 17 17 100 100A-I SN8 20 2 10.0 3.3

HH-821-2' Hulan embryo HF25 18 18 100 100B-1 SN8 20 3 15.0 11. 7

HM-825-1 stem HF25 5 5 100 100Hallm SN8 5 5 100 100

HM-825-1 spermoderm HF25 20 20 100 100A-I SN8 20 20 100 100

HM-825-4 cotyledon HF25 19 19 100 93.0B-2 Hallm SN8 22 8 36.4 19.7

HM-825-4 husk HF25 18 18 100 100B SN8 22 3 136 12 1

HM-826-1 stem HF25 19 19 100 94.7Lmkou SN8 19 3 15.8 7.0

HM-826-1 embryo HF25 18 18 100 98.1A-2 SN8 21 1 4.8 1.6

HF25 2 2 100 100Pmg-Rs SN8 3 3 100 77.8

USA Clark 16 16 100 97 9CK HF25 21 0 0 0

SN8 20 0 0 0

1805

note

one infected plant

different infected

husks on one

infected plant

different infected

husks on one

infected plant

one infected plant

one mfected plant

one infected plant

Proceedmgs of the 7th International Worhng Conference on Stored-product Protectwn - Yolume 2

Table 4. The result of the experiment of seed-transnussion.

infected seed No of infected plant incidence % Isolated site

plant from plant from Isolatedinfected inoculated strainseed healthy seed

cotyledon stem, basal 5stem

basal stem 1

treat- status No. of health Infected health infected healthyment seed seed seed seed seed seed

A complete 7 14 1 4 14 21seed

B separated 10 19 1 5into

spermodermand

cotyledon

Conclusion and Discussion

In 1959, Klein reported the aetiology of phytophthora rootrot, he considered that the seed IS one of the source ofinfection besides the soil, the conclusion 'there IS noevidence that the pathogen IS transmitted by seeds' waswntten in the book Soybean Diseases published by APS,USA. Tills paper identified the SItes where the pathogenexisted, forms of the pathogen m the seed and theprerequisite for seed-bearing. Isolation test showed thatPhytophthora sojae Isolated from any parts of the infectedseed had the same pathogenicity. These conclusions werethe first report and provided scientific baSIS for soybean seedtest for phytophthora root rot.Soybean phytophthora root rot could be carried by seeds,

but It needed to be theoretically clear whether this diseasecould be transmitted by seeds. ThIS paper confirmed thisresult, and cleared up the international distrust of tlus

problem.Oospores of Phytophthora eoiae were produced in

spermoderm only, thereby the standard of seed tests forphytophthora root rot was suggested that only spermodermshould be exammed. Seeds should be Immersed m KGB andspermoderms should be exammed under the microscope.The oospores produced in the seeds have not germmatedyet, this problem needs to be further studied later

References

McGee D. c.. 1990. Soybean DIseases APS S1. Paul,Mmnesota.

Schmittenner A. F., 1985 Plant DIsease, 69, 362 - 368Smc1air ]. B. , 1984. Compendium of Soybean Diseases. APSSt Paul, Minnesota, 104 p.

Zhou Z. B., et aI., 1996, Plant Quarantme, 10, 257-261

1806