Download General research methods on pathogen of potato late blight

Journal of Agricultural University of Hebei
Apr. 2001
General research methods on pathogen of potato late
blight(Phytophthora infestans)
ZHU Jie-hua, ZHANG Zhi- ming, YANG Zhi-hui
( College of Plant Protection. , Agricultural University of Hebei, Baoding 071001, China)
Abstract: It’s known that Phytophthora infestans is very difficult to be isolated and
purified. According to the working experience in the past 8 years, the general research
methods of potato late blight were summarized in this paper, which includes the method of
sample collection of Phytophthora infestans, evaluation of late blight resistance in potato
germplasm.
Key words: Phytophthora infestans; research method; isolate.
CLC number: S 435.32
Document code: A
Late blight is the most serious fungal disease of potatoes that occurs almost anywhere that
pathogens grow. Potato late blight is caused by Phytophthora infestans and it is very
difficult to be isolated and purified. From 1993, the authors began to study potato late
blight, according to the working experience in our laboratory in almost 8 years, the general
research methods on potato late blight were summeried as follows.
1 The method of sample collection of Phytophthora infestans
1.1 Symptons of potato late blight
The leaves, stems and tubers can all be infected by Phytophthora infestans.
Leaves. The earliest symptoms of the disease are often present on lower leaves. They
consist of small, pale to dark green spots that change into brown or black lesions,
depending on the humidity of the air. Lesions begin frequently at leaf tips and margins. The
shape of typical lesion is “V” shape. Under conditions of high humidity and cool
temperature, lesions expand rapidly. A white mildew may be visible at the lower surface of
the leaves. In less than a week, the disease may spread from the first infected leaflet on a
few plants to most plants in a field. Leaves may drop off.
Stems. When the disease is sever, lesions may develop on petioles and stems, where
they expand lengthwise. The colors of the lesions is black. Infected stems are weakened,
and may cause death of plant parts above the lesion.
Tubers. Infected tubers show a superficial and irregular discoloration. The brown
necrotic lesions penetrate from the surface into the tuber tissue. Secondary infections may
often occur in infected tubers and cause soft rot.
1.2 Method of sample collection
In major potato growing areas, the diseased leaf samples were taken in the fields during
occurrence or epidemic of late blight. The leaves with typical symptom ( the lesion was
small and a white mildew was visible) were selected. We cut out the leaves with initially
infected single lesion. The lesion was cut into small pieces(5 – 10 mm2 )at margin of the
lesion, then inoculated potato tuber by inserting a small piece of lesion into cut tuber. The
ZHU Jie-hua General research methods on pathogen of potato late blight(Phytophthora infestans)
cut tuber was sealed with adhesive tape, put into a plastic bag or paper bag and taken back
to the lab. The authors also took the infected leaves from plant, date preserved it in icebox
after covering with paper and plastic bag.
Lesions were sampled randomly from different infected plants within a field. Each
sample should be labeled with place, date of collection and the name of host variety.
2 The method of isolation and purification of Phytophthora infestans
2.1 Isolation of Phytophthora infestans
The inoculated potato tubers were cut into pieces with side length of 5 – 7 mm. Each
piece was put in the moist petri-dish and incubated at 15 – 18ºC. The diseased leaf also
could be cleaned with tap water, cut out a small piece at margin of a lesion and placed on
the tuber slices in moist petri-dish, then incubated at 15 - 18ºC. After 3 days, the pathogen
colony was formed on tubers slices.
2.2 Purification of Phytophthora infestans
2.2.1 Medium (Agar) Biyun bean agar: baiyun bean powder 60 g, V8 juice 100 mL , agar
15 g, CaCO3 1.4 g, dH2 O 1,000 mL
(1). Put 60 g lima bean powder in 500 mL dH2 O, and then cook for 1 hour at 70 – 80ºC
in distilled water.
(2).Filter through four layers of cheesecloth and discard the sediment.
(3).Add 1.4g of CaCO3 to 100 mL V8 juice.
(4).Bring the volume of the combined juice to 1 liter with distilled water.
(5).Add 15 g agar and autoclave at 15 Psi for 15 – 20 minutes.
Rye agar: rye 60 g, agar 20 g, sucrose 20 g, dH2 O 1,000 mL
(1). Soak 60 g rye grains in distilled water for 20 – 30 hours
(2). Boiling the rye grains in 1,000 mL distilled water for 0.5 h.
(3). Filter through 4 layers of cheesecloth and discard the sediment.
(4). Add 15 g agar and 20 g sucrose in the diltrate.
Adjust the filtrate to one liter.
(5) Autoclave at 15 Psi for 15-20 minutes.
2.2.2 Purification of Phytophthora infestans . The medium with antibiotics (rifampicin 20
mg. mL-1 , ampicillin 200 mg. mL-1 , benlate 100 mg. mL-1 ) was poured into sterilized petridish to make plate. The white colony of late blight pathogen was removed from tuber slices
then inoculated on plate medium. After culturing 7 – 10 days at 15 – 18ºC, the pure colony
of Phytophthora infestans was transferred onto slant medium at incubated at 15 – 18ºC in
darkness till full growth, then moved into the incubatorat 10 – 12ºC for maintenance.
3 The identification of Phytophthora infestans
Phytophthora infestans belongs to the class Oomycetes, order Peronosporales , family
Phthiaceae and genus Phytophthora. The vegetation is mycelium characterized by the
absence of cross walls, among which both asexual and sexual reproduction occurs. The
sporangiophores and sporangia emerge at asexual reproduction phase. The sporangia are
lemon shape, measurement of 21 – 38um x 12 – 23 um. Sporangia develop at the end of
these sporangiophores. Oospores are formed at asexual reproduction. When mycelia of
ZHU Jie-hua General research methods on pathogen of potato late blight(Phytophthora infestans)
different mating types of the fungus grow together, one of them may form antheridia and
the other oogonia. The oogonium grows through the antheridium, allowing fertilization.
The fertilized oogonium develops into a thick-walled oospore, while the oospore is orange
red, nearly round-shaped, measurement of 28 – 32 um.
4 Evaluation of late blight resistance in potato germplasm
4.1 Preparation of inoculum
Cultures of Phytophthora infestans were grown on Baiyun bean agar medium in petridishes for 10 – 14 d at 18ºC in darkeness. The sporangia were harvested using 10 mL sterile
water and a cotton swab. The suspensions were filtered through four layers of cheesecloth
to remove mycelial fragments and were adjust to 25, 000 sporangia per milliliter with a
hemacytometer. After the sporangia concentration was determined, the suspension may be
placed at 4ºC until ready to be used (up to 3 hours).
This was also done by inoculating healthy potato slices with Phytophthora infestans,
then incubated at 15 – 18ºC for 5 –7 days. The sporangia were harvested by using 10 mL
sterile water. The suspensions were filtered though four layers of cheesecloth to remove
sediment. The suspensions were placed at 4 – 10ºC. After 2 – 3 hours, the zoospores were
released. The suspensions were filtered again with filter, in which the bore diameter was 12
um to remove sporangia wall. The zoospore suspension were adjusted to 20,000 – 50,000
zoospores per milliliter with a hemacytometer.
4.2 Evaluation (assay) of late blight resistance
1). Inoculated method – inoculation on detached leaves. The number of the 3 – 5 th leaf
from the top of the plant was selected for inoculation. Five leaflets of each compound leaf
were used, so the fifteen leaflets were available. The selected leaflets were placed in a
plastic box with water transportation to the lab. Then the leaflets were placed upside down
in the petri-dish with water agar, inoculated each leaflet with one drop (10 uL) of zoospore
suspension using a Pasteur pipet. The petri-dishes were placed in the incubator at 18ºC with
a twelve hours photo period / 24 hours after inoculation. From the third day after
inoculation, the areas were measured.
2). The estimation of lesion growth rate (LGR) and statistics. From the third day after
inoculations, the lesion area was measured by an electronic instrument. The lesion area
should be measured three times from the third to the fifth day after inoculation. The
maximun length and width were measured. The lesion area was calculated with the
formula: A=1/ 4 r x length x width. According to the lesion area, the lesion was divided
into successful infection and unsuccessful infection. But if the lesion was limited in the
inoculating spot, this infection was considered as unsuccessful infection. If the lesion areas
measured in these three times were all less than inoculation spots, it could be considered as
hypersensitive reaction. Therefore, it was not involved during the calculation of LGR.
Once the lesion area was larger than 16 mm2 among the three times, it was considered as
successful infection. For this successful infection, the radius of the lesion was calculated
based on the lesion area. LGR was figured out according to linear analys is between lesion
radius and time.
References:
ZHU Jie-hua General research methods on pathogen of potato late blight(Phytophthora infestans)
1
HOGSON W A. Laboratory testing of the potato for partial resistance to
Phytophthora infestans. American Potato Journal 38: 259-264.