Dr. Abdul RehmanAssistant Professor, Department of plant pathology, University of Agriculture Faisalabad.

To study the Morphological characterization of Ceratocystis manginecans sp. nov. isolates from Pakistan

In our country export quality mango fruit is produced but unfortunately we are facing with the low production of mango due to the mango sudden death syndrome disease.

Symptoms on trees include gum exudation with severe rotening smell, bark splitting, vascular discoloration with black and white streaks, total or partial wilting of the tree by succeeding browning of

leaves small holes with galleries due to the presence of bark beetle

(Hypochryphalus mangifera Stebbing: Coleoptera Scolytidae) (Al-Adawi 2002; Al-Adawi et. al., 2003).

The fungal isolates recovered from affected mango trees wooden tissues in Pakistan shows distinct as Ceratocystis manginecans from species complex of C. fimbariata sensu stricto and sensu lato.

Sampling:Several mango orchards were visited from Khanewal, Multan, Muzzaffergarh and Rahim Yar Khan during 2010 to 2011. Stem white and black streaked cross sections were collected from the affected mango trees.

Isolation was done by the carrot method (Moller and De Vay, 1968)

Ascospore from ascomatal masses were collected and purified on 2% malt extract agar media (MEA).

All isolates are maintained in Disease diagnostic lab, Department of Plant pathology, University of Agriculture Faisalabad.

Morphological characterization of isolates was done as, The ascomatal bases were globosed,

black (153- ) 192-254 (-281) µm in diameter. Ascomatal necks were dark brown to black becoming lighter at apices

(514- ) 557-635 (-673) µm long, (25- ) 32-42 (-48) µm wide at base, (14- ) 16-22 (-26) µm wide at apices.

Ostiolar hyphae were hyaline and divergent (42- ) 45-59 (-69) µm long.

Ascospores were hyaline, hat-shaped (3-4) µm long, (4-5) µm wide excluding sheath and (7-8) µm wide including sheath.

(Van Wyk, et al., 2007)

Primary cylindrical-shaped hyaline conidia were long (15- ) 23-29 (-33) µm wide (3- ) 4-6 (-6) µm.

Secondary conidia were barrel shaped and hyaline with length (8- ) 9-12 (-12), width (5- ) 5-7 (-8).

Incase of resting spores Chlaymadospores were thick walled, dark brown 11-14 µm long (9-12) µm wide.

(Van Wyk, et al., 2007)

All the isolates are morphologically compared which provides the cause is a fungi named asC. manginecans

Sr. No.

Sample No.

Date of sample

collection

Location of sample collection

Date of isolation

Results

GPS Values In vitro (Ceratocystis manginecans.)

Elevation North East1 26(a) 20-1-11 MZG, Qasba gujrat. 378' 30˚09.273" 70'57.355" 20-1-11 Yes2 27S(a) 20-1-11 MZG, Qasba gujrat 357' 30˚09.286" 70˚57.362" 20-1-11 Yes

Isolates

Sample no.

Place c. manginecans

Morphology

Ascospore Perithecia Conidia ChlaymadosporesW l Beak l Beak w Globosed

ascomaCylindrical Barrel

e.s w.s tip base w W l w l w l26a Muzzafergarh Yes 4 7 4 625 18 38 225 5 29 5 12 9 1227a Muzzafergarh Yes 4 7 3 650 22 28 237 5 20 6 10 10 13

Morphology

26a 27a

Perithecia Beak Tip

conidia

A.spore

Sr. No.

Sample No.

Date of sample

collection

Location of sample collection

Date of isolation

Date of Perithecia

appearGPS Values (Ceratocystis manginecans.)

Flag No. Elevation

North East

3 28 25-3-2011 Talamba, Teh. Mian Channu

Distt. Khanewal.

324 424 30°31.097´

72°13.062´ 25-3-2011 5/4/2011 Yes

4 40 26-3-2011 Keekar wali Basti R.B. Thatha

Qureshi, Muzzafergarh

338 421 29°55.875´

71°10.497´ 26-3-2011 2/4/2011 Yes

Isolates

40

28 Khanewal. Yes 4 7 3 555 25 35 200 5 23 7 12 9 11

40 Muzzafergarh Yes 4 7 4 575 17 35 183 5 29 5 10 9 12

Sample no.

Place c. manginecans Morphology

Ascospore Perithecia Conidia ChlaymadosporesW l Beak l Beak w Globosed

ascomaCylindrical Barrel

e.s w.s tip base w W l w l w l

Morphology

28 40

5 46 26-3-2011 Habibabad, Muzzafergarh

345 327 30°04.550´

71°14.268´ 26-3-2011 2/4/2011 Yes

6 57 27-3-2011 Thana Alpaa, Multan.

358 383 30°19.728´71°31.207´ 27-3-2011 6/4/2011 Yes

Sr. No.

Sample No.

Date of sample

collection

Location of sample collection

Date of isolation

Date of Perithecia appearGPS Values (Ceratocystis

manginecans.)

Flag No. Elevation

North East

Isolates

46 Muzzafergarh Yes 4 6 4 658 18 37 255 5 30 5 10 9 1157 Multan Yes 4 7 4 655 18 40 250 5 29 5 11 9 11

MorphologySample no.

Place c. manginecans

Morphology

Ascospore Perithecia Conidia ChlaymadosporeW l Beak

lBeak w Globosed

ascomaw

Cylindrical Barrel

e.s w.s tip base W l w l w l

46 57

7 58 27-3-2011 Thana Alpaa, Multan.

359 380 30°19.724´ 71°31.227´ 27-3-2011 5/4/2011 Yes

8 59 27-3-2011 Thana Alpaa, Multan.

360 380 30°19.701´ 71°31.251´ 27-3-2011 4/4/2011 Yes

Sr. No.

Sample No.

Date of sample

collection

Location of sample collection

Date of isolation

Date of Perithecia appearGPS Values (Ceratocystis

manginecans.)

Flag No. Elevation

North East

Isolates

58 Multan Yes 5 7 4 650 25 32 230 4 28 6.25 11.25 10 1359 Multan Yes 4.5 7 3 645 17 38 225 4 26 5 12 9 11

MorphologySample no.

Place c. manginecans

Morphology

Ascospore Perithecia Conidia ChlaymadosporeW l Beak l Beak w Globosed

ascomaw

Cylindrical Barrel

e.s w.s tip base W l w l w l

58 59

9 61 27-3-2011 Baber Chock, Multan.

362 349 30°09.793´

71°32.346´ 27-3-2011 7/4/2011 Yes

10 63 27-3-2011 Baber Chock, Multan.

364 373 30°09.904´

71°32.302´ 27-3-2011 5/4/2011 Yes

Sr. No.

Sample No.

Date of sample

collection

Location of sample collection

Date of isolation

Date of Perithecia appearGPS Values (Ceratocystis

manginecans.)

Flag No. Elevation

North East

Isolates

61 Multan Yes 4 7 3 625 22 25 188 5 28 5 10 9 12

63 Multan Yes 4.5 7.5 3 514 24 29 238 5.5 27 6 11 10 14

Sample no.

Place c. manginecans

Morphology

Ascospore Perithecia Conidia ChlaymadosporeW l Beak l Beak w Globosed

ascomaw

Cylindrical Barrel

e.s w.s tip base W l w l w l

Morphology

6163

11 64 27-3-2011 Trri aali bhaini, Multan.

365 336 30°09.718´

71°32.668´ 27-3-2011 5/4/2011 Yes

12 65 27-3-2011 Bhaini, Multan. 366 376 30°10.012´ 71°32.874´ 27-3-2011 5/4/2011 Yes

Sr. No.

Sample No.

Date of sample

collection

Location of sample collection

Date of isolation

Date of Perithecia appearGPS Values (Ceratocystis

manginecans.)

Flag No. Elevation

North East

Isolates

64 Multan Yes 4 7 4 520 25 38 225 5 23 7 12 11 13

65 Multan Yes 5 7 3 263 26 33 238 6 30 5 10 9 11

Sample no.

Place c. manginecans

Morphology

Ascospore Perithecia Conidia ChlaymadosporeW l Beak l Beak w Globosed

ascomaw

Cylindrical Barrel

e.s w.s tip base W l w l w l

Morphology

6465

13 67 28-3-2011 11/8R-Makhdum pur, Teh. Kabeer

wala, Distt. Khanewal

369 412 30°27.610´72°05.816´ 28-3-2011 4/4/2011 Yes

14 68 28-3-2011 11/8R-Makhdum pur, Teh. Kabeer

wala, Distt. Khanewal

370 412 30°27.612´ 72°05.815´ 28-3-2011 6/4/2011 Yes

Sr. No.

Sample No.

Date of sample

collection

Location of sample collection

Date of isolation

Date of Perithecia appearGPS Values (Ceratocystis

manginecans.)

Flag No. Elevation

North East

Isolates

67 Khanewal. Yes 4 7 3.5 650 23 30 230 5 25 5 10 10 12

68 Khanewal. Yes 5 7.5 3 660 24 40 225 5.5 29 5 10 10 13

Sample no.

Place c. manginecans

Morphology

Ascospore Perithecia Conidia ChlaymadosporeW l Beak l Beak w Globosed

ascomaw

Cylindrical Barrel

e.s w.s tip base W l w l w l

Morphology

6768

15 70 28-3-2011 11/8R-Makhdum pur, Teh. Kabeer

wala, Distt. Khanewal

372 425 30°27.595´ 72°05.807´ 28-3-2011 6/4/2011 Yes

16 71 28-3-2011 11/8R-Makhdum pur, Teh. Kabeer

wala, Distt. Khanewal

373 418 30°27.637´

72°05.791´

28-3-2011 4/4/2011 Yes

Sr. No.

Sample No.

Date of sample

collection

Location of sample collection

Date of isolation

Date of Perithecia appearGPS Values (Ceratocystis

manginecans.)Flag No. Elevatio

nNorth East

Isolates

70 Khanewal. Yes 5 7 3 655 23 28 245 4.5 25 7 12 11 14

71 Khanewal. Yes 4.5 7 3 650 24 30 238 5 23 6 11 9 11

Sample no.

Place c. manginecans

Morphology

Ascospore Perithecia Conidia ChlaymadosporeW l Beak l Beak w Globosed

ascomaw

Cylindrical Barrel

e.s w.s tip base W l w l w l

Morphology

70 71

17 72 28-3-2011 11/8R-Makhdum pur, Teh. Kabeer

wala, Distt. Khanewal

374 420 30°27.647´ 72°05.776´ 28-3-2011 4/4/2011 Yes

18 73 28-3-2011 Satara Morr, Teh. & Distt. Khanewal.

375 398 30°30.455´ 72°11.050´ 28-3-2011 4/4/2011 Yes

Sr. No.

Sample No.

Date of sample

collection

Location of sample

collection

Date of isolation

Date of Perithecia appearGPS Values (Ceratocystis

manginecans.)

Flag No. Elevation

North East

Isolates

72 Khanewal. Yes 4 7 3 625 17 35 225 5 22 7.5 12 9 11

73 Khanewal. Yes 5 7 4 650 15 38 240 5 25 7 11.5 9 12

Sample no.

Place c. manginecans

Morphology

Ascospore Perithecia Conidia ChlaymadosporeW l Beak l Beak w Globosed

ascomaw

Cylindrical Barrel

e.s w.s tip base W l w l w l

Morphology

72 73

19 74 28-3-2011 WaryamaAbad, Teh. &

Distt. Khanewal.

376 402 30°30.647´ 72°11.251´ 28-3-2011 5/4/2011 Yes

20 75 28-3-2011 Talamba road khanewal.

377 441 30°30.724´ 72°11.642´ 28-3-2011 4/4/2011 Yes

Sr. No.

Sample No.

Date of sample

collection

Location of sample

collection

Date of isolation

Date of Perithecia

appearGPS Values (Ceratocystis manginecans.)

Flag No.

Elevation North East

Isolates

74 Khanewal. Yes 4.5 7.5 4 550 20 36 235 4 26 6 10 9 1475 Khanewal. Yes 4.5 7.5 4 525 25 25 252 5 33 6 11 10 13

Sample no.

Place c. manginecans

Morphology

Ascospore Perithecia Conidia ChlaymadosporeW l Beak l Beak w Globosed

ascomaw

Cylindrical Barrel

e.s w.s tip base W l w l w l

Morphology

7475

21 76 28-3-2011 Talamba road khanewal.

378 445 30°30.726´ 72°11.645´ 28-3-2011 7/4/2011 Yes

22 87 14-5-2011 Sher Shah (Toll plaza) Multan

D87 368 30°04.367´ 71°18.411´ 14-5-2011 28-5-2011 Yes

IsolatesSr. No.

Sample No.

Date of sample

collection

Location of sample collection

Date of isolation

Date of Perithecia

appearGPS Values (Ceratocystis manginecans.)

Flag No.

Elevation North East

76 Khanewal. Yes 5 7 4 562 27 27 250 5 20 7 12 11 14

87 Multan Yes 4 7 3 538 25 34 250 4 22 6 10 10 12

Sample no.

Place c. manginecans

Morphology

Ascospore Perithecia Conidia ChlaymadosporeW l Beak l Beak w Globosed

ascomaw

Cylindrical Barrel

e.s w.s tip base W l w l w l

Morphology

76 87

23 88 14-5-2011 Sher Shah (Toll plaza) Multan

D88 363 30°04.371´ 71°18.425´ 14-5-2011 25-5-2011 Yes

24 91 14-5-2011 Sunaryanwali, Khan garh,

Muzzafergarh

D91 356 29°55.315´ 71°08.758´ 14-5-2011 29-5-2011 Yes

IsolatesSr. No.

Sample No.

Date of sample

collection

Location of sample collection

Date of isolation

Date of Perithecia

appearGPS Values (Ceratocystis manginecans.)

Flag No.

Elevation North East

88 Multan Yes 5 7.5 4 525 25 33 225 5 29 6 11 12 14

91 Muzzafergarh Yes 5 7.5 4 600 25 38 200 5 30 5 11 11 13

Sample no.

Place c. manginecans

Morphology

Ascospore Perithecia Conidia Chlaymadospore

W l Beak l Beak w Globosed ascoma

w

Cylindrical Barrel

e.s w.s tip base W l w l w l

Morphology

8891

25 92 14-5-2011 Sunaryanwali, Khan garh,

Muzzafergarh

D92 343 29°55.310´ 71°08.757´ 14-5-2011 28-5-2011 Yes

26 95 14-5-2011 Sunaryanwali, Khan Garh,

Muzzafergarh

D95 345 29°55.331´ 71°08.780´ 14-5-2011 3/6/2011 Yes

IsolatesSr. No.

Sample No.

Date of sample

collection

Location of sample

collection

Date of isolation

Date of Perithecia

appearGPS Values (Ceratocystis manginecans.)

Flag No.

Elevation North East

92 Muzzafergarh Yes 4.5 7 3 514 23 35 250 4 20 6 10 11 14

95 Muzzafergarh Yes 4 7 3 575 17 33 253 5 20 5 10 9 12

Sample no.

Place c. manginecans

Morphology

Ascospore Perithecia Conidia ChlaymadosporesW l Beak l Beak w Globosed

ascomaw

Cylindrical Barrel

e.s w.s tip base W l w l w l

Morphology

92 95

27 104 16-5-2011 Union council bhao di

qureshan , RYK.

D104 272 28°36.847´ 70°16.312´ 16-5-2011 3/6/2011 Yes

28 105 16-5-2011 Union council bhao di

qureshan , RYK.

D105 275 28°36.832´ 70°16.313´

16-5-2011 6/6/2011 Yes

IsolatesSr. No.

Sample No.

Date of sample

collection

Location of sample collection

Date of isolation

Date of Perithecia

appearGPS Values (Ceratocystis manginecans.)

Flag No.

Elevation North East

104 Rahim Yar Khan

Yes 5 7.5 3 525 20 25 250 5 23 7 10 10 13

105 Rahim Yar Khan

Yes 4 7 4 520 17 35 183 5 29 6 12 9 11

Sample no.

Place c. manginecans

Morphology

Ascospore Perithecia Conidia ChlaymadosporesW l Beak l Beak w Globosed

ascomaw

Cylindrical Barrel

e.s w.s tip base W l w l w l

Morphology

104 105

29 106 16-5-2011 Moza mohamadAli, RYK.

D106 283 28°39.529´ 70°15.721´ 16-5-2011 6/6/2011 Yes

30 108 16-5-2011 Kurr man singh, road moza shhna

wala, Dhaloon agri frm, RYK.

D108 335 28°36.373´ 70°13.618´ 16-5-2011 6/6/2011 Yes

IsolatesSr. No.

Sample No.

Date of sample

collection

Location of sample collection

Date of isolation

Date of Perithecia

appearGPS Values (Ceratocystis manginecans.)

Flag No.

Elevation North East

106 Rahim Yar Khan

Yes 5 7.5 3 625 23 33 238 6 30 5 10 10 14

108 Rahim Yar Khan

Yes 5 7.5 4 550 20 25 200 5 24 5 12 9 14

Sample no.

Place c. manginecans

Morphology

Ascospore Perithecia Conidia ChlaymadosporesW l Beak l Beak w Globosed

ascomaw

Cylindrical Barrel

e.s w.s tip base W l w l w l

Morphology

106 108

31 109 14-9-2011 Mouza MohmadAli Magssi, RYK.

D109 295 28°37.963´ 70°15.897´

14-9-2011 27-9-2011 Yes

32 110 14-9-2011 Union council Bhao Di

Qureshan, RYK.

D110 293 28°36.716´ 70°17.739´

14-9-2011 29-9-2011 Yes

IsolatesSr. No.

Sample No.

Date of sample

collection

Location of sample collection

Date of isolation

Date of Perithecia

appearGPS Values (Ceratocystis manginecans.)

Flag No.

Elevation North East

109 Rahim Yar Khan

Yes 4 7 3 520 20 25 225 5 22 6 11 10 13

110 Rahim Yar Khan

Yes 4 7 4 550 18 25 250 4 23 6 10 10 14

Sample no.

Place c. manginecans

Morphology

Ascospore Perithecia Conidia ChlaymadosporesW l Beak l Beak w Globosed

ascomaw

Cylindrical Barrel

e.s w.s tip base W l w l w l

Morphology

109 110

33 113 15-9-2011 Basti Bahram Baray Khoo Wali

Mouza Thul Ganga

Qureshan,, RYK.

D113 305 28°39.318´ 70°23.429´

15-9-2011 Yes

34 115 15-9-2011 Abad Pur, RYK. D115 288 28°39.212´ 70°22.542´

15-9-2011 29-9-2011 Yes

IsolatesSr. No.

Sample No.

Date of sample

collection

Location of sample collection

Date of isolation

Date of Perithecia

appearGPS Values (Ceratocystis manginecans.)

Flag No.

Elevation North East

113 Rahim Yar Khan

Yes 4 7.5 3.5 600 20 28 225 5 18 5 10 9 12

115 Rahim Yar Khan

Yes 4 7 4 625 18 30 225 6 20 5 10 11 13

Sample no.

Place c. manginecans

Morphology

Ascospore Perithecia Conidia ChlaymadosporesW l Beak

lBeak w Globosed

ascomaw

Cylindrical Barrel

e.s w.s tip base W l w l w l

Morphology

113115

35 119 16-9-2011 Par Labbay Road , RYK.

D119 277 28°33.914´ 70°15.535´ 16-9-2011 Yes

36 121 17-9-2011 Bosan Farm KLP road, RYK.

D121 295 28°57.664´ 70°08.758´

17-9-2011 27-9-2011 Yes

IsolatesSr. No.

Sample No.

Date of sample

collection

Location of sample collection

Date of isolation

Date of Perithecia

appearGPS Values (Ceratocystis manginecans.)

Flag No.

Elevation North East

119 Rahim Yar Khan

Yes 25 38 12 14

121 Rahim Yar Khan

Yes 5 8 4 675 22 38 250 6 25 6 12 10 12

Sample no.

Place c. manginecans

Morphology

Ascospore Perithecia Conidia ChlaymadosporesW l Beak l Beak w Globosed

ascomaw

Cylindrical

Barrel

e.s w.s tip base W l w l w l

Morphology

119121

37 122 17-9-2011 JDW, Akhtarabad,

RYK.

D122 282 28°31.253´ 70°01.799´

17-9-2011 24-9-2011 Yes

38 125 17-9-2011 Kurr Maan Singh, RYK.

D125 305 28°36.146´ 70°13.675´

17-9-2011 24-9-2011 Yes

IsolatesSr. No.

Sample No.

Date of sample

collection

Location of sample collection

Date of isolation

Date of Perithecia

appearGPS Values (Ceratocystis manginecans.)

Flag No.

Elevation North East

122 Rahim Yar Khan

Yes 4 7 3 675 20 45 275 5 30 7 12 9 13

125 Rahim Yar Khan

Yes 5 7.5 4 550 20 33 270 5 25 6 11 9 12

Sample no.

Place c. manginecans

Morphology

Ascospore Perithecia Conidia ChlaymadosporesW l Beak l Beak w Globosed

ascomaw

Cylindrical Barrel

e.s w.s tip base W l w l w l

Morphology

122 125

39 126 17-9-2011 Kurr Maan Singh, RYK.

D126 299 28°36.405´ 70°13.454´ 17-9-2011 24-9-2011 Yes

40 127 17-9-2011 Kurr Maan Singh, RYK.

D127 298 28°36.412´ 70°13.464´ 17-9-2011 24-9-2011 Yes

IsolatesSr. No.

Sample No.

Date of sample

collection

Location of sample collection

Date of isolation

Date of Perithecia

appearGPS Values (Ceratocystis manginecans.)

Flag No.

Elevation North East

126 Rahim Yar Khan

Yes 4.5 7.5 4 600 15 45 188 4 20 7 11 10 13

127 Rahim Yar Khan

Yes 5 7.5 3 635 15 35 220 5 25 6 12 11 13

Sample no.

Place c. manginecans

Morphology

Ascospore Perithecia Conidia ChlaymadosporesW l Beak l Beak w Globosed

ascomaw

Cylindrical Barrel

e.s w.s tip base W l w l w l

Morphology

126 127

In our study the range of morphological characters are as follows which is almost fall in the prescribed range of Van Wyk.

The ascomatal bases were globosed, black (153- ) 183-275 (-281) µm in diameter.

Ascomatal necks were dark brown to black becoming lighter at apices (514- ) 514-650 (-673) µm long, (25- ) 25-45 (-48) µm wide at base, (14- ) 15-26 (-26) µm wide at apices.

Ostiolar hyphae were hyaline and divergent (42- ) 45-62 (-69) µm long.

Ascospores were hyaline, hat-shaped (3-4) µm long, (4-5) µm wide excluding sheath and (7-8) µm wide including sheath.

Primary cylindrical-shaped hyaline conidia were long (15- ) 18-32 (-33) µm wide (3- ) 4-6 (-6) µm.

Secondary conidia were barrel shaped and hyaline with length (8- ) 9-12 (-12), width (5- ) 5-7 (-8).

Incase of resting spores Chlaymadospores were thick walled, dark brown 11-14 µm long (9-12) µm wide.

Further identification will be done on the bases of molecular characterization which is under progress.

Special thanks to Punjab Agriculture Research Board for funding this project.