Breeding of Psidium Species for Root Knot Nematode Resistance in Malaysia A.R. Milan Horticulture Research Centre, MARDI P.O. Box 12301, General Post Office 50774 Kuala Lumpur Malaysia Keywords: Guava, Psidium guajava L., root knot nematode, Meloidogyne incognita Abstract

The root knot nematode (Meloidogyne incognita) was reported to cause severe damage in guava cultivation in Malaysia. Several long term approaches were taken to find sustainable control for the root knot nematode through breeding. The approaches include protocol development to screen nematode resistance in Psidium species, genetic resource establishment in the field, screening of accessions against the nematode, identification of resistant rootstock and hybridization of resistant accessions with commercial cultivars. Protocol developed was used to screen more than twenty accessions against the nematode, and three accessions were found to be potential. These resistant accessions will be further evaluated for rootstocks. Hybridization among Psidium species was initiated to study the genetic compatibility among the species. Thirteen parental combinations were successfully hybridized and more than 2000 F1 interspecific hybrid seeds were sown and will be further evaluated for resistance to root knot nematodes. INTRODUCTION

Guava is one of the fruit types that were given priority in commercial cultivation in Malaysia. The area planted with guava increased from 856 ha in 1986 to 1809.2 ha in 2000. Production of guava in 2000 was 11,674 mt for fresh consumption at value of RM11 million at the price of RM 1,000/mt (Table 1). This was generated from production area of 1,097 ha at the yield of 14.4 mt/ha for fresh (Anon, 2003). Expansion of guava cultivation is good because the crop was included in one of the priorities fruits under cultivation policy of Ministry of Agriculture especially in the Malaysian Third National Agriculture Policy.

The most serious problem facing the guava industry in Malaysia is the root-knot nematode (Meloidogyne incognita). The attack was widespread across the country and all commonly grown guava varieties were reported to be susceptible (Abdul Karim and Yuen, 1994). Abdul Karim et al. (2002) screened 28 nematode-free seedlings of guava cultivar and showed that nematode gave high root galling index and high death rate for the guava cultivars evaluated except in red strawberry guava (P. littorale var. longipes) which showed apparent tolerance. Cuadra and Quincosa (1982) who screened 5 Psidium species in Cuba found that Costa Rican guava (P. friedrichsthalianum) was highly resistant to Meloidogyne spp.

In nematode study, resistance plant is defined as plants that cannot prevent the parasite’s entrance but is able to prevent, restrict or retard its development. Resistance reactions are manifested when some factors in the host upsets the relationship between the host and nematode. Resistance is expressed by larvae failing to develop into adults, prolongation of the developmental cycle, larva death with root tissue, alteration of sex ratio toward maleness, or larva egress soon after penetration (Fassuliotis, 1979). In this work, the product is guava plants that can withstand the ravages of nematode attack, to enable them to grow and produce well.

Previous works done by Abdul Karim et al. (2002) shows that local accessions have potential resistance to the root knot nematode and detailed studies have to be done to identify accessions systematically. The accessions can be multiplied and used as rootstock with

61Proc. Ist IS on Guava Eds. G. Singh et al. Acta Hort. 735, ISHS 2007

commercial clones and can also be used in breeding programme with specific objectives. The breeding objectives of guava improvement programme in Malaysia can be

summarised as development of guava plants with resistance to nematode, high yielding especially seedless varieties, flower and fruiting manipulation to optimize and continuous off season production and also to improve production system with less use of paper bags to reduce labour cost.

This paper will report some of the progress made in long term approach to find sustainable control for this nematode through breeding approach. Various stages that were taken to achieve these objectives consist of: (i) Development of protocol to screen nematode resistance in guava species. (ii). Establishment of genetic resources materials in the field. (iii). Screening a range of Psidium species against the nematode. (iv) Using promising resistance as rootstock. (v) Hybridisation using accessions identified as resistance as parent with commercial clones. MATERIAL AND METHODS

There are several stages in these long term programme of breeding guava for resistance to root knot nematode in Malaysia. Nematode Screening Procedure

The detail screening protocol was developed and described by Abdul Rahman et al. (2005). Guava seeds were germinated in germinating tray. Sterilised top soil was filled in the polybag (17 cm × 7 cm). After 60 days, the seedlings were transplanted into polybag. Seedlings to be screened were prepared in four replicates. Once the plants were well-established, it was inoculated in each polybag with 5000 eggs of a known species of Melodogyne incognita. After around 60 days, the test was terminated by harvesting the plant. Plant damage was assessed for galls index by observation and counted the total eggs extracted from egg masses in the roots of the plant as in Table 2 (Taylor and Sasser, 1978). Nematode reproduction was assessed and host efficiency was calculated using Oostenbrink’s reproduction factor: R = Pf/Pi, where Pi was the initial inoculum levels (5000 eggs) and Pf was the final egg formed in the root. Canto-Saenz (1983) host suitability designations were assigned on the basis of the scale proposed by Oostenbrink (1966) as in Table 3 and Table 4. Genetic Resources Collection, Conservation and Evaluation 1. Long Term Genetic Conservation. A total of 24 accessions were already established in germplasm plot at MARDI Serdang. These consist of Department of Agriculture, Malaysia registered clones as well as selected clones by individual farmers, nurseryman and include other than P. gujava species. This collection will become a genepool based for future research with various objectives including selection for root knot nematode. 2. Genetic Collection and Evaluation for Root Knot Nematode. A total of 180 accessions were collected from local and overseas. In this collection, locally natural grown P. guajava and others Psidium species fruits were collected and germinated in the glasshouse. Those that were germinated were given selection pressure to select against root knot nematode using method developed by Abdul Rahman et al. (2005). Those accessions that showed potential resistance were planted in the germplasm plot for long term conservation and evaluation. Selection of Accessions for Rootstock

The potential accessions that showed tolerance or resistance to root knot nematode were multiplied and raised in the glasshouse for rootstock selection. The selected seedlings were used as rootstock and will be grafted with scion from established commercials clones. All grafted seedlings were evaluated in nematode hot spot area in the field.

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Hybridisation of Selected Accessions Available accessions or clones that show potential resistance were selected as

parents in the breeding programme for nematode resistance. Those plants that already flowered were used in the hybridisation programme. Selected flower buds were monitored and tagged when it sepals started to crack at around 1-2 days before actual crossing can be done. Flower bud that was matured had size around 1-2 cm diameter with glossy skin and some sepal splitting. The flower buds were pollinated 24-36 hours later after sepal splitting. Pollination was done from 8.00 to 12.00 hours. Pollen source was collected from flowers of different accessions that already opened and produced pollen. After pollination, the flowers were covered with small bag made from soft tracing paper or muslin cloth to prevent foreign pollen contamination. After 3-5 days, the bag was removed especially if the flower bud starts to form small fruits. When the fruit size reached 2-3 cm diameter, the fruit was sprayed with a mixture of fungicide and pesticide then it was covered with paper or plastic bag to control against fruit fly and any diseases. The fruit was harvested when its skin colour turn yellowish. That seeds were collected and germinated in the sand-filled tray. The established seedlings were transferred into the field for further evaluation. RESULTS AND DISCUSSION Screening Protocol

Results show that there was a wide variation in term of plants response to the root-knot nematode attack. That means the protocol was able to differentiate guava species and can be used to evaluate the response of individual accession to root knot nematode attack. Analysis of variance shows that all parameters studies were significantly different among the accessions (Table 5). Table 6 shows that using the proposed protocols, the mean and range of the parameters studied were significantly different that contribute to high coefficient of variation for parameter of total root weight (37.65), total egg masses in the root (47.72), total egg in the root (45.93), final egg produced (81.91) and host efficiency (82.04). CV value was low only for gall index parameter. Analysis of means for all the parameter studied shows that there is a significant difference between all the accessions as shown in Table 7. Based on all results, only 3 out of 19 accessions have the potential resistance to root knot nematode as shown in Table 8. The protocol developed was successful in quantifying the ability of individual accession to respond to the nematode attack. The results gave the guideline to select the most potential accession that was resistant to nematode character.

Genetic Resources Establishment

Germplasm collection in MARDI was started in early 1990 when the fruit commodity was given priority by Ministry of Agriculture. But after 1995, the priorities were changed and research activities for guava was not done comprehensively. Still small land holders continue to plant guava using their own selection or introduced materials. Genetic resources were conserved by various agencies as shown in Table 9. In 2005, 180 accessions of naturally grown guava were collected for root knot nematode screening. Also, 24 accessions that have selected characters and established clones were collected and conserved in MARDI for long term objectives are shown in Table 10.

Selection for Rootstock

Results showed that commercial clone GU8, that was used as control in the test was the most vulnerable to nematode attack because it had the highest Rf value (16.78) as shown in Table 8. This was expected as GU8 was planted using vegetative propagation (marcott) and was known to be susceptible to the root knot nematode. However, observation of the Rf value showed that plants propagated by seeds were less vulnerable to the nematode attack as compared to the plant propagated by vegetative means. These results supported the proposal to use resistant rootstock for the propagation of guava.

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Only 3 accessions namely P. guajava acc. B-12, P. littoralle var. longipes and P. arayan showed potential resistance to nematode. According to Canto-Saenz (1983), the three accessions were designated as hypersusceptible where their Rf values are 0.88, 0.15 and 0.15 respectively (Table 8). Hypersusceptible means that the root attacked by the nematode will be damaged but the nematode will not survive or multiply inside the root of the plants.

Evaluation of the growth character showed that P. littoralle var. longipes and P. arayan were not suitable for rootstock. P. littoralle var. longipes had slow growth and spreading growth habit. The species did not produce upright single stem for efficient propagation. P. arayan has the potential resistance to nematode and observation showed that plant growth habit is too slow and their graft compatibility is not yet tested and still questionable because of inter-species compatibility. Only P. guajava acc. B-12 has the potential to control the root knot nematode and graft-compatible with commercial clone of guava because they are from the same species.

Hybridisation of Guava

Earlier guava breeding in MARDI started with some selection in 6th Malaysia Plan for fresh dessert guavas. The planting materials were mostly imported from Thailand and germinated in Serdang station. Healthy seedlings were planted in Kemaman station and early evalution produced ten good selected plants. Ten selected seedlings were planted and further evaluated in Jeram Pasu station. From that evaluation, two green-dessert varieties namely JP1 and JP2 was developed for the industry. However, both varieties are not resistant to nematodes (Norlia, 1994). After year 2000, the objective of breeding programme was modified to solve the problems in guava cultivation that was to produce new guava varieties with resistance to root-knot nematode and high yield for fresh and processing. The initial hybridisation result was show in Table 11. To date, more than 2000 F1 seedlings were germinated and the plants were in the nursery stages for further evaluation. CONCLUSION

The resistance and susceptibility of the accessions was measured by gall index (GI) as an indicator for plant damage. Host efficiency (R factor) as an indicator for nematode reproduction of the tested accessions. This system (which used nematode reproduction to quantify host efficiency and extent of galling or yield loss to quantify plant damage) can give an indication of nematode resistance. Preliminary results of this study showed that the protocol developed was successful in screening the resistance of Psidium species to the root knot nematode. The results also showed that all P. guajava accessions, except accession B-12, were susceptible to the nematode, with commercial clone (GU8) was the most susceptible. P. longipes and P. arayan showed potential resistant characters to the root-knot nematode but their potential as rootstock for guava cultivation is still questionable. The results showed that P. guajava acc. B-12 had the potential control to the nematode and could be used as a rootstock for guava commercial clones. A total of more than 2000 F1 seedlings were produced in the hybridisation programme and the F1 seedlings are in the nursery stages for further evaluation. ACKNOWLEDGEMENTS

This study was supported by IRPA grant 01-03-03-0144-EA001 to MARDI from Ministry of Science, Technology and Innovation Malaysia. The author thanks Director General of MARDI and Director of Horticulture Research Centre MARDI for permission to present this paper. Also thanks to Mrs Najah Yahaya, Mr. Zaidun Bilal Mat and Mrs. Umikalsum Mohd Bahari for their technical assistance. Literature Cited Abd Rahman, M., Abdul Karim, S. and Najah, Y. 2005. Development of preliminary

screening protocol for root-knot nematode resistance in Psidium species. Proceeding

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of MARDI Horticulture Research Centre Technical Meeting. 26-29 June 2005, Penang Malaysia: 21-29

Abdul Karim S. and Yuen P.M. 1994. Permasaalahan dan pengurusan nematode puru akar pada tanaman jambu batu. Teknologi Buah-buahan MARDI. 10:13-17

Abdul Karim, S., Yuen, P.M. and Norlia, Y. 2002. Challenges in the integrated management of the root-knot nematode on guava. Proceeding Mesyuarat Teknikal Pusat HR Port Dickson: 27

Anon. (2003). Hectareage of guava by year, Peninsular Malaysia http://agrolink.moa.my/doa/BI/Statistics/jambu_batu.html

Canto-Saenz, M. 1983. The nature of resistance to Meloidogyne incognita (Kofoid and White, 1919) Chitwood 1949. In. C.C. Carter. (ed). Proc. Third Res. and Plann. Conf. on Root-knot Nematodes Meloidohyne spp. March 22-26 1982. International Meloidogyne Project, Lima Peru: 160-165

Cuadra R. and Quincosa A. 1982. Potential of different Psidium gujava species as sources for resistance of guava to Meloidogyne. Ciencias-de-la-Agricultura 13:19-26

Fassuliotis G. 1979. Plant breeding for root knot nematode resistance. In. Lamberti F and Taylor CE (eds). Root-knot nematodes (Meloidogyne species) - Syatematic, Biology and Control. Academic Press: 425-453

Norlia, Y. 1994. Klon-klon jambu batu yang berpotensi. Teknologi Buah-buahan 10: 9-12 [in Malay].

Oostenbrink, M. 1966. Major characteristics of the relation between nematodes and plants. Meded. Landbouwhogesch. Wageningen 66 (4). 46 pp.

Sasser, J.N., Carter, C.C. and Hartman, K.M. 1984. Standardization of host suitability stucies and reporting of resistance to root-knot nematodes. Raleigh, North Carolina, USA. 7p.

Taylor, A.L. and Sasser J.N. 1978 Biology, identification and control of root-knot nematodes (Meloidogyne spp.) Coop. Pub. Dept. Plant Pathol. North Carolina State University and US. Agency Int. Dev. Raleigh, N.C. 111 pp.

Tables Table 1. Area and production of guava in Malaysia (2000).

State Total area (ha) Production area (ha) Production (mt)

Johor 713.5 581.1 4,888.8 Kedah 56.7 39.9 72.0 Kelantan 38.5 14.5 92.7 Melaka 49.4 49.4 1,235.0 Negeri Sembilan 22.8 7 32.3 Pahang 47.7 27.0 362.1 Perak 342.7 304.7 3,812.1 Perlis 2.0 2.0 3.0 Pulau Pinang 20.5 14.9 77.1 Selangor 49.8 46.5 794.6 Terengganu 26.4 10.1 304.5 Pen. Malaysia 1,370.0 1,097.1 11,674.2 Sabah 183.2 - - Sarawak 256.0 - - Total 1,809.2 1,097 11,674.2 Source: Department of Agriculture (2003)

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Table 2. Taylor and Sasser’s rating scale for the presence of root-knot nematode galls or egg masses on roots.

Number of galls or egg masses Gall index (GI) or egg mass index (EI)

0 0 1-2 1 3-10 2 11-30 3 31-100 4 100+ 5

Source: Taylor and Sasser (1978) Table 3. Host suitability designations proposed by Canto-Saenz.

Damage to plant Nematodes reproduction on host Statistically significant Insignificant Efficient Susceptible Tolerant Inefficient Hypersusceptible Resistance

Source: Canto-Saenz (1983) Table 4. Quantitative scheme for assignment of Canto-Saenz’s host suitability

(resistance).

Plant damage (gall index) Host efficiency (R factor) Degree of resistance (DR) designation

≤ 2 ≤ 1 Resistant ≤ 2 > 1 Tolerant > 2 ≤ 1 Hypersusceptible > 2 > 1 Susceptible

Source: Canto-Saenz (1983) Table 5. Means square of analysis of variance for six parameters of 19 Psidium accessions

tested for nematode response.

Source df

Total root

weight (g)

Total egg masses in the root

Total egg in the root

Final egg produce (Pf)

Host efficiency

(Rf)

Gall Index (GI)

Accessions 18 136.97** 17184.63** 8125.62** 1402121558.39** 56.14** 1.60**Error 57 4.13 3019.58 2388.78 233469598.28 9.36 0.32 Total 75

** = significantly different at p<0.01

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Table 6. Summary of the parameters studied.

Parameter Mean Range C.V (%)

Total root weight 5.39 1.2 – 33.4 37.65 Total egg masses in the root 115.15 2 – 385 47.72 Total egg in the root 106.42 5.3 – 303.7 45.93 Final egg produce (Pf) 18654.70 38 – 145350 81.91 Host efficiency (Rf) 3.73 0 – 29.1 82.04 Gall Index (GI) 4.45 2 – 5 12.64

Table 7. Means comparison of six different parameter on 19 Psidium accessions.

Accession

Total root

weight (g)

Total egg masses in the

root

Total egg in the root

Final egg produce after inoculation

(Pf)

Host efficiency

(Rf)

Gall Index (GI)

W-1 4.05 de 205.75 ab 179.60 a 35020 bc 7.00 bc 5.00 a W-2 3.10 e 152.25 bcd 152.93 abcd 22319 bcd 4.48 bcd 5.00 a W-3 2.95 e 180.50 bc 88.58 bcdef 16015 bcd 3.20 bcd 5.00 a J-1 3.60 de 123.50 bcdef 167.43 ab 22050 bcd 4.38 bcd 5.00 a C-1 1.60 e 70.25 defgh 104.80 abcdef 6549 d 1.33 d 4.25 abc C-2 4.3 de 137.00 bcde 116.33 abcde 16339 bcd 3.28 bcd 4.75 ab B-1 2.48 e 57.00 efgh 96.58 bcdef 6269 d 1.28 d 3.75 cd B-3 3.23 e 120.75 bcdef 161.43 abc 18255 bcd 3.65 bcd 4.75 ab B-5 3.05 e 95.25 cdefgh 164.65 abc 16467 bcd 3.30 bcd 4.50 abc B-8 6.55 cd 180.50 bc 117.25 abcde 21485 bcd 4.30 bcd 5.0 a B-12 4.75 de 105.25 cdef 43.43 ef 4570 d 0.88 d 4.50 abc B-13 4.45 de 102.00 cdefgh 93.00 bcdef 10338 cd 2.08 cd 4.75ab N-2 2.80 e 81.00 defgh 56.90 ef 5818 d 1.15 d 4.25 abc N-3 2.95 e 100.00 cdefgh 120.58 abcde 12117 bcd 2.43 bcd 4.50 abc N-4 3.05 e 120.00 bcdef 84.25 cdef 18621 bcd 3.73 bcd 4.00 bcd P. longipes 7.9 c 13.00 h 50.23 ef 710 d 0.15 d 2.75 e P. arayan 2.65 e 27.25 gh 25.90 f 874 d 0.15 d 3.25 de J. Gerap 11.45 b 279.25 a 123.90 abcde 36 780 b 7.34 b 4.50 abc GU-8 27.63 a 37.35 fgh 74.23 def 83845 a 16.78 a 5.00 a

Mean values with the same letter in the same column are not significantly different at p< 0.05 according to the DMRT.

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Table 8. Preliminary result of screening of Psidium accessions for root-knot knot nematode.

Accession Initial egg inoculum

(Pi)

Final egg produce after inoculation

(Pf)

Host efficiency

(Rf)

Gall Index (GI)

Degree of resistance

designation

W-1 5000 35020 7.00 5.00 susceptible W-2 5000 22319 4.48 5.00 susceptible W-3 5000 16015 3.20 5.00 susceptible J-1 5000 22050 4.38 5.00 susceptible C-1 5000 6549 1.33 4.25 susceptible C-2 5000 16339 3.28 4.75 susceptible B-1 5000 6269 1.28 3.75 susceptible B-3 5000 18255 3.65 4.75 susceptible B-5 5000 16467 3.30 4.50 susceptible B-8 5000 21485 4.30 5.00 susceptible B-12 5000 4570 0.88 4.50 hypersusceptible B-13 5000 10338 2.08 4.75 susceptible N-2 5000 5818 1.15 4.25 susceptible N-3 5000 12117 2.43 4.50 susceptible N-4 5000 18621 3.73 4.00 susceptible P. longipes 5000 710 0.15 2.75 hypersusceptible P. arayan 5000 874 0.15 3.25 hypersusceptible J. Gerap 5000 36 780 7.34 4.50 susceptible GU-8 5000 83845 16.78 5.00 susceptible Table 9. Numbers of germplasm conserved by different agencies in Malaysia.

No Agency No. of accessions

1 Department of Agriculture, Lekir 20 2

ARS Lagud Seberang Tenom, Sabah 14

3 Golden Hope Plantation, Sitiawan 6 4 ARC Tarat, Sarawak 19 5 MARDI Serdang 22

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Table 10. Accesions conserved in MARDI germplasm Serdang.

No. Accession no. Acessions name

1 MG 1 Thai maroon 2 MG 2 Seedless Bidor 3 MG 3 Seedless Kajang 4 MG 4 Seedless Mantin 5 MG 5 Seedless irregular Bidor 6 MG 6 Seedless elongate Bidor 7 MG 7 Seedless round Bidor 8 MG 8 Yellow strawberry 9 MG 9 Red strawberry 10 MG 10 Guava Philippines 11 MG 11 Guava Vietnam 12 MG 12 Kampuchea (GU8) 13 MG 13 Lee Pear 14 MG14 Yong Peng Pink 15 MG 15 Beaumont Kluang 16 MG 16 Lee Apple 17 MG 17 Montein 18 MG 18 Sali Yak 19 MG 19 Psidium acutangulum 20 MG 20 Psidium arayan 21 MG 21 Beaumont Serdang 22 MG 22 Seedless Serdang 23 MG 23 Guava Gerap 24 MG24 Psidium guinensis

Table 11. Preliminary hybridisation result done in 2004.

Female Male Fruit no. Maturity days Ave. fruit weight (g) No. of seeds

MG 1 MG 3 1 115 490 188 MG1 MG4 1 116 330 160 MG1 MG10 1 109 330 374 MG1 MG10 2 116 440 103 MG1 MG10 3 114 410 160 MG1 MG10 4 124 480 182 MG10 MG1 1 105 155 99 MG15 MG1 1 107 140 53 MG15 MG1 2 109 160 67 MG15 MG18 1 116 275 179 MG18 MG1 1 116 400 305 MG18 MG17 1 107 630 295 MG18 MG17 2 107 570 148

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