SEED-TO-PLANT TRANSMISSION OF PSEUDOMONAS SYRINGAE PATHOVARS

IN SOYBEAN (GLYCINE MAX)

STUDENT: NDONDO JOSEPH

CANDIDATE: N0110859W

DEGREE: B.SC.. HONS APP BIO & BIOCHEM

FIELD OF STUDY: PLANT PATHOLOGY

SUPERVISORS: MR. K. MUSHONGA

INTRODUCTION (JUSTIFICATION)

• To reduce the impact of severe consequence plant disease outbreaks so as to

maintain a high soybean crop production.

• To assist and improve in plant disease diagnosis and other plant pathology

research in the country by providing up-to-date scientific data.

• To understand seed-to-seedling transmission as it is the most critical point of plant

infection as 90% of the world food crops including soybean (Glycine max) are

propagated by seed (Maude., 1996) and many recycle seeds (farmer-saved seeds).

OBJECTIVES

• To determine seedling emergence of various soybean (glycine max)

cultivar seeds infected by pseudomonas syringae pv. glycinea

• To determine and compare the incidence and severity of disease as a

result of seed to seedling transmission of pseudomonas syringae pv.

glycinea in various soybean (glycine max) cultivars.

• To determine the seed-to-seedling transmission efficiency of

pseudomonas syringae pv. glycinea in various soybean (glycine max)

cultivars.

LITERATURE REVIEW

•Pseudomonas syringae is ranked first in the list of top

10 plant pathogenic bacteria, as reviewed by the journal

of molecular plant pathology in 2012 (Scholthof et al.,

2011).

• It is a gram-negative, rod-shaped, obligate aerobic and

motile by one or more polar flagella

• Soybean is a leguminous vegetable of the pea family that grows in

tropical, subtropical, and temperate climates.

• Soybean (Glycine max) Bacterial blight disease caused by

Pseudomonas syringae pv. glycinea impacts heavily on soybean

production and has been reported to cause significant yield losses

ranging from 4% to as high as 40% under extreme conditions.(Mishra

and Krishna ., 2001).

• It has been established that both commercial and small-scale farmers

in Zimbabwe recycle seeds every 2-4 years (Kananji et al., 2013)

SUB-SAHARAN AFRICA SOYBEAN PRODUCTION AND PROCESSING RANKINGS

(FAO., 2011; USDA., 2012)

METHODOLOGY

BACTERIAL STRAIN CONFIRMATION

• Three Pseudomonas syringae strains isolated from farmer-saved seeds from Guruve, Chivhu and Harare and characterized, were confirmed on 3 selective media: Medium M71, Medium D4, Modified Sucrose Peptone (MSP) and 1 semi-selective one; Milk Tween Agar.

COLLECTION OF SEED SAMPLES AND BREAKING SEED DORMANCY

•Three different farmer-saved soybean (Glycine max) landraces were

sourced from local farmers in Chivhu, Guruve and Harare. In addition,

SEEDCO PVT LTD kindly provided five commercial soybean

(Glycine max) varieties namely: Sequel, Santa, SC Saga, Squire and

Status.

SEED INOCULATION WITH PATHOGEN

• Samples of 120 seeds each per each variety/landrace of soybean (glycine max) were directly inoculated by soaking in bacterial inoculum of 106 to 107 colony forming units (CFU)/ml.

• Seeds were then air-dried for 1 hour in a biosafety cabinet at room temperature.

PLANTING, GERMINATION AND SYMPTOM DEVELOPMENT

• Seeds were randomly sampled from the inoculated lot immediately

after drying and planted in germination packets.

• Control plants were not inoculated with pathogen but soaked in broth

only were also planted.

• Seedling emergence was assessed 4-5 d after planting and seedlings

were left to grow to allow symptom development.

• Preliminary symptoms of Bacterial blight disease such as blight

spots, yellow-to-brown necrosis on cotyledons and failure of leaf

development were observed and recorded.

CONFIRMATION OF INFECTION & QUANTIFICATION OF BACTERIA

• Infection was confirmed by culturing surface disinfected plant tissue

(cotyledon and leaves) of both symptomatic and symptomless

seedlings on Selective Medium Modified Sucrose Peptone., Mohan

and Schaad., 1987.

• Antibiotics: Ampicillin, Kanamycin and an antifungal were

incorporated.

• On MSP after 4 days, Pseudomonas. syringae pv. colonies are

circular, raised globose, glistening and light yellow Colonies on MSP

medium with a less dense centre. The medium around the colony

turns light yellow after 3 days.

RESULTS

Iso late 1 Iso late 2 Iso late 30

5

10

15

20

25

30

35

40

45

50

SEEDLING EMERGENCE AFTER 4-5 DAYS

ChivhuHarareGuruveSignalSquireSequelStatusSaga

SOYBEAN VARIETY

% S

EED

LIN

G E

MER

GEN

CE

Iso late 1 Iso late 2 Iso late 30%

10%

20%

30%

40%

50%

60%

70%

80%

90%

Disease Incidence at cotyledon stage

GuruveSignalSquire SequelStatusSaga

variety

DIS

EA

SE IN

CID

EN

CE

PATHOLOGICAL RESULTS

STATUS ISOLATE 2

Symptoms on cotyledon

SEQUEL ISOLATE 2SAGA ISOLATE 2

CONTROL

QUANTIFICATION OF BACTERIA

SQUIRE ISOLATE 1

SQUIRE ISOLATE 1

STATUS ISOLATE 1

STATUS ISOLATE 1

10-2 dilution on MSP

psg

HARARE ISOLATE 1

HARARE ISOLATE 1

SAGA ISOLATE 2

SAGA ISOLATE 2

10-2 dilution on MSP

psg

Symptomless plant

SEQUEL ISOLATE 3 psg

10-2 on MSP

DISCUSSION

• All three Pseudomonas syringae pv. isolates severely crippled

germination rates especially in the Harare, Chivhu and Guruve

landraces. This is likely due to their “farmer-saved” nature.

• From the preliminary results, seed-to-plant transmission of all

Pseudomonas syringae pv. isolates was positive in all soybean

varieties however the soybean variety SAGA showed relatively lower

disease incidence levels.

• Preliminary results also showed that Pseudomonas syringae pv. Had

been successfully transmitted from seed to plant despite the

symptomless plant tissue

• From the results there is a correlation between the severity of

symptoms and the MSP counts recovered from the plant tissue

• Seed germination averaged a mere 13% in all varieties. the lower

temperatures involved in the present study may have enhanced the

lower germination rates.

RECOMMENDATION

• The use of PCR and ELISA to detect and quantify Pseudomonas syringae pv. In symptomatic and

symptomless plant tissue

• For ELISA: 104 bacteria in 1 mL can be detected (Barzic and Trigalet, 1982) and it is a fast, simple and

cheap method.