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16. Recent developments in early in vitro screening for resistance against migratory endoparasitic nematodes in Musa - Elsen A., D. de Waele


Laboratory of Tropical Crop Improvement
Katholieke Universiteit Leuven
Kasteelpark Arenberg 13
3001 Heverlee
Belgium

Abstract

Of the nematodes that parasitise bananas throughout the world, Radopholus similis and Pratylenchus coffeae are the most damaging, causing severe yield losses in crops grown commercially and for local consumption. Chemical control is currently the method used most to manage these nematodes, although nematocides are highly toxic and expensive. Therefore, nematode control through genetic improvement of banana is widely encouraged. Many Musa genotypes have been screened to find resistance against these root pathogens. Screening for nematode resistance is time consuming, because it must be carried out both under field and greenhouse conditions. In vitro screening could facilitate and speed up the incorporation of genetic nematode control into bananas. However, an in vitro screening method requires aseptic nematode cultures. In this paper, the development of aseptic cultures of R. similis and P. coffeae and an in vitro screening method will be discussed.

Alfalfa callus on modified White's medium has proved to be a good aseptic culture system for both R. similis and P. coffeae. Although reproduction is significantly lower than in carrot disc cultures, this system has many advantages. Not only are the nematodes cultured under completely aseptic conditions, but this system is also less labour intensive and offers a more continuous inoculum production. In addition, culturing on alfalfa callus does not alter the pathogenicity of R. similis and P. coffeae. Both R. similis and P. coffeae could infect and reproduce on the roots of in vitro grown 'Grande Naine' plants. For both nematodes, necrotic lesions were observed in the roots within 2-3 weeks after inoculation. The in vitro screening procedure was optimised by determining the optimal initial inoculum and incubation period. Finally, the in vitro screening procedure was validated by testing the reproduction of R. similis in vitro on six different Musa genotypes with a known host response to R. similis. Except for cv. Yangambi Km5, the host response of these genotypes under in vitro conditions corresponded to their host response under greenhouse or field conditions. The susceptibility of 'Grande Naine', 'Gros Michel' and 'Cachaco' was confirmed as well as the resistance of 'Pisang Jari Buaya' and 'SH-3142'.

1. INTRODUCTION

Among nematodes practising bananas throughout the world, the migratory endoparasitic lesion-nematodes Radopholus similis and Pratylenchus coffeae are the most damaging [1], causing severe yield losses in crops grown commercially and for local consumption [2]. Chemical control is currently the method used most to manage these nematodes, although nematocides are highly toxic and expensive. Therefore, nematode control through genetic improvement of banana is widely encouraged. Many Musa genotypes have been screened to find resistance against these root pathogens. Screening for nematode resistance is time consuming because it must be carried out under both field and greenhouse conditions. In vitro screening could facilitate and speed up incorporation of genetic nematode control into bananas. However a useful in vitro screening method requires aseptic nematode cultures. In addition to screening of existing Musa germplasm and improved hybrids from the breeding programs, an in vitro screening method could also be used for the evaluation of anti-nematode proteins for their effectiveness against these lesion-nematodes. Arabidopsis thaliana is used as a model system for genetic studies. Anti-nematode proteins are easily expressed in transgenic lines. Sijmons et al. [3] reported that A. thaliana was a good host for migratory and sedentary endoparasitic nematodes. Therefore, A. thaliana was suggested as a new model system for plant-parasitic nematodes.

The most commonly used substrate for in vitro culture of R. similis and P. coffeae is carrot discs [4]. Although reproduction is very high, this culture technique makes in vitro studies difficult because the nematodes are easily contaminated. At K.U.Leuven, a study was initiated with three objectives: (a) to establish an aseptic culture system for these nematodes; (b) to determine whether the nematodes can infect and reproduce in vitro on banana plantlets and A. thaliana; and (c) to develop and validate an in vitro screening procedure for Musa germplasm.

2. DEVELOPMENT OF AN ASEPTIC CULTURE METHOD FOR RADOPHOLUS SIMILIS AND PRATYLENCHUS COFFEAE

2.1. Methodology

2.1.1. Preparation of alfalfa callus

Alfalfa seeds (Medicago sativa L.) were sterilized with a 15 min soak in concentrated H2SO4, followed by four rinses with sterile, distilled water; a 15 min soak in HgCl2 (1000 p.p.m. in 30% ethanol); followed again by four rinses with sterile, distilled water [5]. Sterile 4-day-old alfalfa seedlings, produced from these seeds on plates of check agar (10 g sucrose, 2 g yeast agar, 10 g agar, 1000 ml water), were placed on slants. These slants were prepared from 14 ml aliquots of White's medium [6] modified by adding 0.2 p.p.m. a-NAA and 2 p.p.m. 2,4-D. Seven to ten days later, after allowing the callus to develop, the calli were transferred to Petri dishes containing the same medium.

2.1.2. Initiation of nematode cultures on alfalfa callus

The R. similis population from Uganda, previously cultured on carrot discs, was surface-sterilized for 2 min in 0.01% HgCl2, followed by two rinses with sterile, distilled water. With a sterile micropipette, 25 females were inoculated onto each alfalfa callus. The Petri dishes were incubated at 28 ± 0.5°C in the dark, allowing the nematodes to feed and reproduce on the callus. After 5 weeks, the nematodes began moving from the callus. To maintain stock cultures of the sterile R. similis population, fresh alfalfa calli were infected with a small piece of infected callus containing an undetermined number of R. similis. After two subcultures, the nematodes were placed on PDA (potato dextrose agar) and NA (nutrient agar) to test for bacterial and fungal contamination.

2.1.3. Preparation of nematode inoculum

To extract the nematodes, the callus was chopped and put on a sterile 70 µm-pore sieve. The sieve was placed on a sterile watch glass containing sterilized water. Within 48 h, the living nematodes had migrated through the sieve into the water. Prior to inoculation, the nematodes were collected from the bottom of the watch glass. The extraction process was carried out under sterile conditions at room temperature.

2.2. Radopholus similis

2.2.1. Comparison of alfalfa callus with carrot disc as a substrate for culturing Radopholus similis

Alfalfa calli were established in Petri dishes containing 20 ml modified White's medium. Each callus was inoculated with 25 female R. similis extracted from callus stock cultures. Fresh carrot discs were prepared and inoculated with 25 female R. similis from the same Ugandan population, but maintained on carrot discs [7]. Each treatment was replicated ten times. The alfalfa calli and the carrot discs were incubated at 28 ± 0.5°C in the dark. After 5 weeks, the nematodes were extracted using a Baermann funnel for the calli and a maceration-sieving technique for the carrot discs [8, 9]. Numbers of nematodes were determined by counting one 6 ml aliquot of a homogenized 150 ml suspension.

Radopholus similis reproduced well on both alfalfa callus and carrot disc, with a 137- and 379-fold increase of the initial population, respectively (Table 1). Taking into account that the amount of tissue available to the nematodes was similar in both treatments, carrot disc was a significantly better substrate (P £ 0.05) than alfalfa callus. The R. similis population cultured on alfalfa callus tested negative for bacterial and fungal contamination on PDA and NA.

Table 1 Reproduction of Radopholus similis (Ugandan population) on alfalfa callus and carrot disc 5 weeks after inoculation with 25 females, incubated at 28 ± 0.5°C in the dark

Substrate

Total final population

Rr a

Alfalfa callus

3433 a

137 a

Carrot disc

10893 b

379 b

Data are means of 10 replicates. Means in the same column followed by the same letter are not different (P £ 0.05) according to the Tukey test. Data were log(x + 1) transformed prior to statistical analysis.

a Rr = Reproduction ratio.

2.2.2. Reproduction of Radopholus similis on alfalfa callus as a function of time

Alfalfa calli were established in Petri dishes containing 20 ml modified White's medium. Each callus was inoculated with 25 female R. similis extracted from alfalfa callus stock cultures. The calli were incubated at 28 ± 0.5°C in the dark. Six, 9 and 12 weeks after inoculation, eight Petri dishes were analysed by extracting nematodes from the callus and the medium separately, using a maceration-sieving technique. Numbers of juveniles, females, and males were determined by counting three 2 ml aliquots taken from a 50 ml homogenized suspension.

The number of nematodes extracted from the alfalfa callus and the modified White's medium increased over time (Table 2). Within 6 weeks, the life cycle was completed, as indicated by the recovery of 220 females per Petri dish and the presence of males. At that time the reproduction ratio was 26.3. The population continued increasing 119- and 223-fold 9 and 12 weeks after inoculation, respectively, which indicates that alfalfa callus cultured on White's medium is a good host for R. similis. The proportion of females and males at all vermiform stages, counted in the callus and the medium was 33% and 2% at 6 weeks, 38% and 8% at 9 weeks, and 42% and 7% at 12 weeks after inoculation, respectively. All vermiform developmental stages were extracted from the callus and the medium. Toward the end of the experiment more nematodes were moving into the medium, and many nematode clusters were observed on the modified White's medium. The percentage of nematodes recovered from the medium, as a proportion of the total population in the Petri dishes, increased from 15% at 6 weeks after inoculation to 68% at 12 weeks after inoculation.

Table 2 Reproduction of Radopholus similis (Ugandan population) on alfalfa callus 6, 9, and 12 weeks after inoculation with 25 females, incubated at 28 £ 0.5°C in the dark


Nematodes in callus

Nematodes in medium



Time (weeks)

Juveniles

Females

Males

Total

Juveniles

Females

Males

Total

Pf a

Rr b

6

383

160

13

556

38

60

3

101

657 ac

26.3 a

9

1246

654

123

2023

363

491

104

958

2981 b

119.2 b

12

908

746

143

1797

1971

1591

223

3784

5581 c

223.2 c

a Pf, Final population including juveniles, females and males extracted from callus and medium.

b Rr, Reproduction ratio = P f/P i = Final population/Initial population.

Data are means of 8 replicates. Means in the same column followed by the same letter are not different (P £ 0.05) according to the Tukey test. Data were log(x+1) transformed prior to statistical analysis.

2.2.3. Pathogenicity of Radopholus similis cultured on alfalfa callus

In vitro propagated 'Grande Naine' (Musa AAA, Cavendish group) tissue culture plants were used. After propagation, regeneration, and rooting [10], the in vitro propagated plantlets were transplanted into 1-litre plastic pots filled with a 2:1 mixture of autoclaved peat and quartz sand. The pots were maintained in a greenhouse at an ambient temperature of 20-27°C with a 12 h photoperiod. To adapt to the greenhouse conditions, the plants were kept for 2 weeks under a plastic cover, which was gradually opened during the following 2 weeks. The pots were irrigated as needed and fertilized every 3 weeks. Eight weeks after planting, each plant was inoculated with 1000 vermiform nematodes. Eight plants were inoculated with R. similis cultured on carrot discs and another eight were inoculated with alfalfa callus-cultured nematodes. The plants were harvested 8 weeks after inoculation. The percentage of necrotic root tissue was assessed for each plant. In addition, the nematode population density per root system and per gram fresh root weight was determined. A maceration-sieving technique was used to extract nematodes. For the extraction, a subsample of 15 g of fresh roots was taken, and the nematodes in a 6 ml aliquot taken from a homogenized 150 ml suspension were counted.

On the 'Grande Naine' plantlets, the R. similis population cultured on alfalfa callus proved to be as pathogenic as the population cultured on carrot discs. No significant differences (P £ 0.05) in damage or reproduction were observed between the two populations (Table 3).

Table 3 Test for pathogenicity of Radopholus similis cultured on alfalfa callus and carrot disc on 'Grande Naine' in the greenhouse. Nematode numbers 8 weeks after inoculation with 1,000 vermiforms.


Root weight (g)

Root necrosis (%)

R. similis in total root system

R. similis per gram fresh roota

'Grande Naine' inoculated with R. similis cultured on alfalfa callus

113 a

15 a

10929 a

90 a

'Grande Naine' inoculated with R. similis cultured on carrot disc

129 a

9 a

5726 a

55 a

Data are means of 8 replicates. Means in the same column followed by the same letter are not different (p £ 0.05) according to the Tukey test.

a Data were log(x+1) transformed prior to statistical analysis.

2.3. Pratylenchus coffeae

2.3.1. Comparison of alfalfa callus with carrot disc as a substrate for culturing Pratylenchus coffeae

Alfalfa calli were established in Petri dishes containing 20 ml modified White's medium. Each callus was inoculated with 25 female P. coffeae, extracted from callus stock cultures. Fresh carrot discs were prepared and inoculated with 25 female P. coffeae from the same Ghanaian population but maintained on carrot discs [7]. Each treatment was replicated ten times. The alfalfa calli and the carrot discs were incubated at 28 ± 0.5°C in the dark. Eight weeks after inoculation, the reproduction in both substrates was compared by extracting the nematodes from the tissues and the medium, using a maceration-sieving technique. Numbers of nematodes were determined by counting one 6 ml aliquot of a homogenised 150 ml suspension. The numbers of nematodes were determined as previously described for R. similis in Section 2.2.2.

On both substrates, good reproduction of P. coffeae was observed 8 weeks after inoculation. In the carrot discs, the reproduction was significantly better (Pf[35] = 6815) than in alfalfa calli (Pf = 2287) (Table 4). The P. coffeae population cultured on alfalfa callus tested negative for bacterial and fungal contamination on PDA and NA.

Table 4 Reproduction of Pratylenchus coffeae (Ghanaian population) on alfalfa callus and carrot disc 8 weeks after inoculation with 25 females, incubated at 28 ± 0.5°C in the dark

Substrate

Total final population

Rr a

Alfalfa callus

2287 a

92 a

Carrot disc

6815 b

303 b

Data are means of 10 replicates. Means in the same column followed by the same letter are not different (P £ 0.05) according to the Tukey test. Data were log(x + 1) transformed prior to statistical analysis.

a Rr, Reproduction ratio.

2.3.2. Reproduction of P. coffeae as a function of time

Alfalfa calli were inoculated with 25 female P. coffeae, extracted from alfalfa callus stock cultures. The calli were incubated at 28 ± 0.5°C in the dark. Eight, 10, 12 and 14 weeks after inoculation eight calli were analysed for nematode reproduction. The nematodes were extracted from the tissue and the medium separately, using maceration-sieving technique.

The total nematode population increased over time (Table 5). Eight weeks after inoculation, the life cycle was completed and good reproduction was already observed, with a Rr[36] of 95. The alfalfa callus tissue is a good host for P. coffeae, as the Rr increased to 214 and 319 at 10 and 12 weeks after inoculation, respectively. Towards 14 weeks, however, reproduction was slowing down with a Rr of 234. Probably the alfalfa callus became limiting for nematode reproduction. The proportion of females and males to all vermiform stages, counted in the callus and the medium, was 31% and 20% at 8 weeks, 30% and 18% at 10 weeks, 33% and 20% at 12 weeks, and 25% and 10% at 14 weeks after inoculation, respectively. All vermiform developmental stages were extracted from both callus tissue and medium. Already after 10 weeks more and more nematodes were moving towards the medium.

Table 5 Reproduction of Pratylenchus coffeae (Ghanaian population) on alfalfa callus 8, 10, 12 and 14 weeks after inoculation with 25 females, incubated at 28 ± 0.5°C in the dark


Nematodes in callus

Nematodes in medium



Time (weeks)

Juveniles

Females

Males

Total

Juveniles

Females

Males

Total

Pf a

Rr b

8

1061

643

375

2079

90

105

109

304

2383 a

95 a

10

1076

686

329

2091

1019

924

652

2595

5356 b

214 b

12

2457

1619

757

4833

1295

1035

805

3135

7968 b

319 c

14

1629

628

348

2605

1848

815

575

3238

5843 b

234 bc

a Pf = Final population including juveniles, females and males extracted from callus and medium.

b Rr = Reproduction ratio = P f/P i = Final population/Initial population.

Data are means of 7 replicates. Means in the same column followed by the same letter are not different (P £ 0.05) according to the Tukey test. Data were log(x+1) transformed prior to statistical analysis.

2.3.3. Pathogenicity of Pratylenchus coffeae cultured on alfalfa callus

The same methodology was used as previously described for R. similis in Section 2.2.3. Eight weeks after planting, the 'Grande Naine' plants were inoculated 1000 vermiform P. coffeae. Eight plants were inoculated with P. coffeae cultured on carrot discs and another eight were inoculated with nematodes cultured on alfalfa callus. The plants were harvested 10 weeks after inoculation. Root necrosis and nematode population densities were determined as described for R. similis (Section 2.2.3.).

On 'Grande Naine' plantlets, the P. coffeae population (from Ghana) cultured on alfalfa callus proved to be as pathogenic as the population cultured on carrot disc. No differences (P £ 0.05) in damage potential and nematode reproduction were observed between the two populations (Table 6). In addition, no differences in root weight were observed.

Table 6 Test for pathogenicity of Pratylenchus coffeae cultured on alfalfa callus and carrot disc, on 'Grande Naine' in the greenhouse (analysis 10 weeks after inoculation with 1000 vermiforms)


Root weight (g)

Root necrosis (%)

R. similis in total root system

R. similis per gram fresh root

'Grande Naine' inoculated with P. coffeae cultured on alfalfa callus

53

11

9475

175

'Grande Naine' inoculated with P. coffeae cultured on carrot disc

59

11

6257

110


n.s.

n.s.

n.s.

n.s.

Data are means of eight replicates. Data were log(x+1) transformed prior to statistical analysis.

2.4. Conclusion

Alfalfa callus has proved to be a good substrate for aseptic culturing of different migratory plant-parasitic nematodes, including R. similis and Pratylenchus spp. [11-13]. Our results confirmed this, although a different growth medium was used instead of Krusberg's medium. For the production of Pratylenchus penetrans, the modified White's medium (containing 0.2 p.p.m. a-NAA and 2 p.p.m. 2,4-D) used in our experiments proved to be as good as Krusberg's medium [7].

Although alfalfa callus is a poorer host than carrot disc, it has important advantages. First, the nematodes feed on sterile tissue and appear to be free of bacteria and fungi (after testing on PDA and NA). Second, this culture system needs to be subcultured less frequently and the nematodes are more easily available. Finally, the maintenance of the nematode culture is less intensive-intensive, since it is sufficient to transfer a piece of infected callus or medium to a fresh callus to initiate a fresh culture [11]. In alfalfa callus, the population can reach the same level as in carrot disc [14], although a longer incubation time is needed. The different extraction techniques used in the first experiment may have influenced the results.

Although alfalfa callus has been used previously to culture R. similis, the pathogenicity of the populations cultured on alfalfa callus was never tested. The pathogenicity of other nematode species has been reported to be unaltered after culturing under aseptic conditions. This was reported for Pratylenchus penetrans propagated on alfalfa callus [15], and Heterodera schachtii propagated on transformed beet [16]. Our study confirmed this for the first time for R. similis after culturing on alfalfa callus, using susceptible banana plants in the greenhouse.

3. IN VITRO INFECTION OF 'GRANDE NAINE' AND A. THALIANA BY RADOPHOLUS SIMILIS AND PRATYLENCHUS COFFEAE

3.1. Radopholus similis

3.1.1. Infection and reproduction on 'Grande Naine'

Aseptic shoots of the Musa genotype Grande Naine (AAA, Cavendish group) were transferred to jars containing MS (Murashige and Skoog) rooting medium [10]. The jars, containing two plantlets each, were incubated at 28 ± 0.5°C with a 16 h light/8 h dark cycle. Three weeks later, ten jars were inoculated with 50 female R. similis extracted from alfalfa callus. Mature females were collected individually with a sterile micropipette and placed in a drop of sterile water on the MS medium near the shoots. Eight weeks after inoculation, the nematodes were extracted from the roots and the medium separately, using a maceration-sieving technique. Numbers of juveniles, females, and males were counted in one 6 ml aliquot taken from a 70 ml homogenized suspension.

Table 7 Reproduction of Radopholus similis (Ugandan population) and Pratylenchus coffeae (Ghanaian population) on in vitro plants of 'Grande Naine' (Musa AAA) 8 and 10 weeks, respectively after inoculation with 50 females incubated at 28 ± 0.5°C (16-8 hours light regime)


Juveniles

Females

Males

Total

Pf a

Rr b

R. similis







nematodes in roots

4798

187

128

5,113

6,282

126

in medium

975

146

48

1,169



P. coffeae







nematodes in roots

568

200

147

915

1396

28

in medium

182

126

173

481



a Pf, Final population including juveniles, females and males extracted from callus and medium.

b Rr, Reproduction ratio = Pf/Pi = final population /initial population.

All data are means of eight replicates.

The in vitro grown 'Grande Naine' plantlets were a good host for R. similis: the nematode population increased 126-fold in 8 weeks, reaching on average 6282 juveniles, females and males (Table 7). The nematodes were able to penetrate and reproduce in the roots, since 81% of the total population was recovered from the root system. All vermiform developmental stages were extracted from the roots as well as from the medium. The proportion of females and males counted in the roots and the medium was 4% and 3% in the roots and 12% and 4% in the medium, respectively. Necrotic lesions were observed on the in vitro roots (Figure 1).

3.1.2. Infection and reproduction on A. thaliana

Seeds of A. thaliana (Colombia 0 ecotype) were surface-sterilized with a 2 min soak in 95% ethanol, followed by a 12 min soak in sterile water with 5% NaOCl and 0.1% Tween, and three rinses with sterile distilled water. The sterilized seeds were sown on germination medium [17]. Ten-day-old seedlings were transferred to and arranged on a thin layer of Knop medium [3]. Petri dishes, each containing three seedlings, were placed slightly tilted to promote unidirectional root growth. After growth for 10 more days at 22 ± 0.5°C (16 h-light/8 h-dark cycle), the roots were inoculated with 20 female R. similis per root system (60 females per Petri dish). The Petri dishes were incubated at 28 ± 0.5°C with a 16 h-light/8 h-dark cycle. Ten weeks after inoculation, the nematodes were extracted using a Baermann funnel. Numbers of juveniles, females, and males were counted in three 2 ml aliquots taken from a homogenous 25 ml suspension.

Figure 1 Necrotic lesions on in vitro roots of the susceptible Musa genotype Grande Naine, caused by Radopholus similis.

Figure 2 Radopholus similis in the roots of Arabidopsis thaliana grown under in vitro conditions. Stained with acid fuchsin.

Radopholus similis could successfully penetrate and develop in A. thaliana under monoxenic conditions (Figure 2). Ten weeks after inoculation, the population reached 975 individuals (Table 8). The recovery of 367 females and the presence of males indicated that the life cycle was completed. All vermiform developmental stages were observed in the medium prior to extraction. Lesions on A. thaliana were observed as localized yellowing of the cortical tissue.

Table 8 Reproduction of Radopholus similis (Ugandan population) and Pratylenchus coffeae (Ghanaian population) on in vitro Arabidopsis thaliana, 10 and 8 weeks, respectively, after inoculation with 60 females incubated at 28 ± 0.5°C (16/8 hours light/dark regime)


Juveniles

Females

Males

Pf a

Rr b

R. similis

441

367

167

975

16.3

P. coffeae

376

219

120

715

11.9

a Pf, Final population including juveniles, females and males extracted from callus and medium.

b Rr, Reproduction ratio = Pf/Pi = final population /initial population.

All data are means of eight replicates.

3.2. Pratylenchus coffeae

3.2.1. Infection and reproduction on 'Grande Naine (see Section 3.1.1.)

Aseptic shoots of the Musa genotype 'Grande Naine' were prepared and inoculated with 50 female P. coffeae extracted from alfalfa callus, using the same method as described for R. similis above (Section 3.1.1.). Ten weeks after inoculation, nematode reproduction was determined by extracting and counting the nematodes separately from the in vitro roots and the medium, using the method described for R. similis (Section 3.1.1.).

The 'Grande Naine' plantlets grown in vitro were a good host for P. coffeae; the nematode population increased 28-fold in 10 weeks, reaching on average a total population of 1396 (Table 7). The nematodes could penetrate and reproduce in the roots, since 66% of the total population was recovered from the roots. All vermiform developmental stages were recovered from both roots and medium. The proportion of females and males was 22% and 16% in the roots, and 26% and 36% in the medium, respectively. Necrotic lesions were observed on the in vitro roots. Overall, the reproduction was lower than that of R. similis (Section 3.1.1.).

3.2.2. Infection and reproduction on A. thaliana

The same method as described for R. similis (Section 3.1.2.) was used. The plantlets were inoculated with 60 female P. coffeae. Eight weeks after inoculation, the nematodes were extracted using a Baermann funnel. Numbers of juveniles, females and males were counted in three 2 ml aliquots taken from a homogenous 25 ml suspension.

As for R. similis, P. coffeae could successfully penetrate and develop in A. thaliana roots under monoxenic conditions (Table 8). Eight weeks after inoculation, the life cycle was completed as indicated by the recovery of 219 females and the presence of males. The final population reached 715 living vermiforms. All vermiform developmental stages were observed in the medium prior extraction. Lesions on A. thaliana were observed as localized yellowing of the cortex tissue.

3.3. Conclusion

Based on the good reproduction of R. similis and P. coffeae on in vitro grown 'Grande Naine' plantlets and the necrosis observed in the root systems, this in vitro system could be further developed to identify variations in resistance within Musa germplasm at a very early stage. Preliminary results had already indicated that resistance to R. similis could be identified to some extent in plantlets growing in rooting medium under in vitro conditions [18]. To obtain a reproducible method, it is important to include a highly susceptible genotype as a reference. In the past, 'Grande Naine' was used in greenhouse screening experiments as a susceptible reference genotype [19]. According to Mateille [20], in vitro systems are unsuitable for screening banana genotypes for resistance to R. similis because the in vitro plantlets are too sensitive to reveal variations in resistance. However, our study demonstrates that if the period prior to analysis is not too long and the container is not too small, allowing extensive root growth, variations in resistance might be revealed. For P. coffeae, no further information is available.

Sijmons et al. [3] established A. thaliana as a model system for plant-parasitic nematodes. It was suggested that expressing gene products detrimental to nematodes in this plant might rapidly lead to engineering new genotypes with increased resistance to nematodes. For R. similis and P. coffeae, two important nematodes practising bananas, no information was available on whether A. thaliana could be used as model host plant. In our study, penetration and reproduction were confirmed on bananas for the first time, and an in vitro evaluation system with A. thaliana was further optimised for these tropical endoparasitic nematodes. Two important modifications were made to the in vitro evaluation system: (a) after transfer from germination medium to Knop medium, a 10 day period of culture allowed the plants to develop a branched root system; and (b) after inoculation, the Petri dishes were incubated at 28°C. This temperature is more optimal for the reproduction of R. similis and P. coffeae [21], and does not restrict growth of the A. thaliana plantlets.

4. IN VITRO SCREENING AGAINST RADOPHOLUS SIMILIS AND PRATYLENCHUS COFFEAE

Four experiments were carried out. In the first experiment, different incubation times were tested. Three, 7, 10, 12 and 15 weeks after inoculation with 50 female R. similis eight replicates were analysed (i.e. the development and increase of the nematode population were determined). For the second experiment, different inocula were tested. The plants were inoculated with 5, 10, 25, 50 and 100 female R. similis per jar. Eight weeks after inoculation the plants were analysed. In the third and fourth experiment different Musa genotypes with a known host plant response to R. similis were tested. The plants were inoculated with 25 or 50 female R. similis per jar. All plants were analysed 8 weeks after inoculation.

4.1. Optimisation

In the first experiment, banana plantlets were analysed at different times to determine the optimal duration of a screening experiment, i.e. long enough to have reproduction, but not so long that the food source becomes depleted (this means the roots are still able to grow) (Table 9). After 3 weeks, the initial nematode population had penetrated the Musa roots and reproduced. The populations recovered from the roots and the medium increased with time. At first, the majority of the nematodes were in the roots, but after 15 weeks most of the nematodes were in the medium. This indicates that the food source (i.e. the roots) is becoming depleted. During the whole experiment, in the roots as well as in the medium, the majority of the nematode population consisted of juveniles, while the male proportion was the smallest.

Root necrosis was observed in 80% of the plants 3 weeks after inoculation, while all plants had necrotic areas in the root tissue after 7 weeks. Based on nematode reproduction, the proportions of nematodes in the roots and the medium, and the occurrence of damage, the optimal time for analysis is between 7 and 12 weeks. Therefore, in the following experiments the plantlets were analysed at 8 weeks after inoculation.

The number of nematodes recovered from the roots and medium of banana plantlets inoculated with different initial nematode populations are listed in Table 10. Penetration of the roots was observed for all inoculum densities, and for all densities nematodes were recovered from the roots. The life cycle was completed and reproduction was observed. However, significantly higher numbers of nematodes (P £ 0.05) were recovered from roots and medium for inoculum densities of 25 females or more, indicating that inoculum densities of 25, 50 or 100 female R. similis are optimal and equally suited for in vitro screening. In addition, at the end of the experiment (i.e. 8 weeks after inoculation) necrosis was observed on the roots of the in vitro plantlets at all inoculum densities. As the inoculum density increased, the percentage of necrotic tissue in the roots increased as well. At the end of the experiment all plants were still vigorous, without any sign of nutrient or water deficiency. Based on the results from the previous experiments, an in vitro screening procedure was designed with a nematode inoculum consisting of 25 or 50 female R. similis and an incubation time of 8 weeks.

4.2. Confirmation

Table 11 presents the in vitro reproduction of R. similis on four Musa genotypes with known host plant response. Radopholus similis could reproduce on all genotypes. However, the reproduction on the genotype 'Pisang Jari Buaya' was significantly lower than on the other three genotypes. No significant differences in susceptibility to R. similis were found among the three genotypes 'Grande Naine', 'Gros Michel' and 'Yangambi Km5'.

Table 9 Mean nematode population densities of R. similis (Ugandan population) in the roots of in vitro grown 'Grande Naine' and the medium, at different times after inoculation with 50 females

Time (weeks) (n)

Roots

Medium

Pfb

Rrc

juveniles

females

males

total

juveniles

females

males

total

3 (10)

198 a
(67-425)a

35 a
(0-108)

19 a
(0-83)

252 a
(75-558)

48 a
(0-92)

4 a
(0-17)

17 a
(0-33)

68 a
(17-125)

321 a
(92-650)

6.4

7 (10)

2229 b
(158-4233)

360 b
(33-825)

194 b
(8-458)

2783 b
(258-5042)

1141 b
(50-2533)

710 b
(25-1792)

114 b
(8-333)

1965 b
(83-4658)

4748 b
(908-9608)

95.0

10 (10)

3113 bc
(558-9283)

294 b
(33-675)

138 b
(8-375)

3545 b
(603-10325)

1590 b
(325-4733)

675 b
(50-2450)

124 b
(25-292)

2389 b
(603-7108)

5934 b
(1233-17433)

118.7

12 (10)

4246 bc
(392-11392)

635 bc
(75-1992)

384 b
(42-1183)

5265 bc
(542-14567)

2292 bc
(192-5833)

2167 bc
(142-5683)

130 b
(25-200)

4588 bc
(358-11708)

9853 bc
(900-26275)

197.1

15 (10)

9438 c
(2133-24309)

1998 c
(492-5242)

752 c
(117-2733)

12188 c
(2875-32283)

9808 c
(558-42800)

6784 c
(158-23916)

788 c
(50-2625)

17380 c
(767-69342)

29567 c
(3642-92150)

591.3

Data were log(x+1) transformed prior statistical analysis. Means in the same column followed by the same letter do not differ according the Tukey test (P £ 0.05).

a(minimum-maximum), b Pf = final population in roots and medium, c Rr = Pf / PI (with PI = 50 females).

Table 10 Mean nematode population densities of R. similis (Ugandan population) in the roots of in vitro grown 'Grande Naine' and the medium after 8 weeks, using different inoculum densities (PI)

Inoculum density (n)

Roots

Medium

Pfb

Rrc

juveniles

females

males

total

juveniles

females

males

total

5 EE (11)

62 a
(0-550)a

9 a
(0-83)

8 ab
(0-50)

79 a
(0-683)

96 a
(0-546)

7 a
(0-58)

3 a
(0-33)

106 a
(0-638)

185 a
(0-1321)

37.0

10 EE (11)

52 a
(0-433)

9 a
(0-50)

0 ab
(0-0)

61 a
(0-483)

41 a
(0-350)

7 a
(0-63)

2 a
(0-8)

50 a
(0-421)

111 a
(0-904)

1.1

25 EE (11)

1568 b
(50-5775)

209 b
(0-650))

105 c
(0-450)

1882 b
(50-6875)

505 b
(33-1158)

78 b
(0-175)

21 b
(0-92)

604 b
(33-1425)

2486 b
(83-7458)

99.4

50 EE (11)

870 b
(0-4200)

52 ab
(0-175)

43 bc
(0-250)

965 b
(0-4400)

286 ab
(0-1192)

124 b
(0-633)

27 ab
(0-158)

437 b
(0-1983)

1402 b
(0-6383)

28.0

100 EE (10)

1715 b
(100-3075)

272b
(0-700)

100 c
(0-300)

2087 b
(100-3425)

299 b
(25-583)

186 b
(17-608)

32 b
(0-100)

517 b
(42-1292)

2604 b
(142-4317)

26.0

Data were log(x+1) transformed prior statistical analysis. Means in the same column followed by the same letter do not differ according the Tukey test (P £ 0.05).

a (minimum-maximum), b Pf = final population in roots and medium, c Rr = Pf / PI.

The same experiment was repeated, and some more Musa genotypes were included (Table 12). In general, nematode reproduction was higher than in the previous experiment. 'Cachaco' (Musa ABB, Bluggoe type) appeared to be as susceptible as the susceptible reference genotype 'Grande Naine', while 'SH-3142' proved to be as resistant as 'Pisang Jari Buaya'. In this experiment, the susceptibility of 'Gros Michel' was reconfirmed, while 'Yangambi Km 5' had an intermediate resistance status, neither differing from the resistant genotypes nor from the susceptible ones. For all the genotypes tested, most of the nematodes were recovered from the roots (between 61 and 88%). This indicates that neither the incubation time nor the inoculum density were limiting conditions. The lowest proportion of juveniles was recovered from the roots of the two resistant genotypes, 'Pisang Jari Buaya' and 'SH-3142'.

4.3. Conclusion

A procedure for in vitro screening for nematode resistance in Musa spp. has been set up by determining the optimal incubation time and the optimal nematode inoculum, and confirmed by the host plant response of a selection of Musa genotypes with known host plant response. The optimisation of incubation time and initial inoculum is very important because all plants, susceptible and less susceptible ones, will be equally infected if the conditions become limiting for plant growth and thus for nematode reproduction [20]. Under in vitro conditions the root growth and root mass are limited by the container. If incubation time is too long and/or if the initial inoculum is too high, the differences in resistance and susceptibility might be eliminated, especially if absolute resistance (i.e. no reproduction) is very rare, as in the case of Musa [22].

For the initial inoculum, the optimum was determined between 25 and 100 females, because for those inocula levels good reproduction was observed (with the majority of the nematode population in the roots) and necrotic areas were clearly visible in the in vitro roots. The optimal incubation time was set at 7 to 12 weeks for the same reasons as mentioned above. For practical reasons, and based on these results, a procedure was set up with an initial inoculum of 25 to 50 females and an incubation time of 8 weeks (selected in parallel with the procedure for rapid screening for nematode resistance in the greenhouse [23]). Stoffelen [24] had already studied the reproduction of R. similis on carrot discs in relation to time. The results are comparable, although a different nematode population was used and the nematodes were cultured on carrot disc, instead of alfalfa callus. However, the substrate used for nematode culturing (alfalfa callus or carrot disc) does not influence the reproductive capacity of the R. similis population [25]. Based on their pathogenicity, the Ugandan population [26] used in this experiment is similar to the population from Ivory Coast used by Stoffelen [24]. Both populations have a high reproductive fitness.

In our study only two genotypes showed resistance to R. similis under in vitro conditions, Pisang Jari Buaya and SH-3142. The resistance of these two genotypes has been reported previously under greenhouse conditions as well as under field conditions [18,23,27-30]. The type of initial planting material (corm-derived or tissue-culture-derived plants) did not influence the resistance [18]. The susceptibility of 'Grande Naine' and 'Cachaco' confirmed the previous results in the greenhouse and in the field [18,23,27,29-32]. It was reported that the Bluggoe-type, like 'Cachaco', is as susceptible or even more susceptible than the Cavendish-type, such as 'Grande Naine' [23,32], while the Gros Michel-type has been reported as less susceptible than the Cavendish type [18]. However, these findings were not confirmed in our in vitro screening experiments. For 'Yangambi Km5', the partial resistance or resistance as described by many [18,23,27,31] was not confirmed under in vitro conditions.

Table 11 Reproduction of R. similis (Cuban population) on four Musa genotypes with known host plant response to R. similis, grown under in vitro conditions, 8 weeks after inoculation with 25 females per replicate

Musa genotype (n)

Total in roots

Total in medium

Pfb

Rr c

Pisang Jari Buaya (9)

57 a
(0-240)a

37 a
(0-220)

93 a
(0-390)

3.7

Gros Michel (10)

249 ab
(20-590)

120 ab
(20-230)

369 b
(40-740)

14.8

Yangambi Km 5 (9)

864 ab
(10-2270)

231 b
(70-630)

1096 b
(180-2680)

43.8

Grande Naine (4)

940 b
(480-1420)

363 bc
(200-510)

1303 b
(680-1930)

52.1

Data were log(x+1) transformed prior analysis. Means followed by the same letter do not differ according to Tukey method (P £ 0.05).

a (minimum-maximum), b Pf = final population in roots and medium, c Rr = Pf / PI (with PI = 25 females)

Table 12 Reproduction of R. similis (Ugandan population) on six Musa genotypes with known host plant response, grown under in vitro conditions, 8 weeks after inoculation with 50 females per replicate

Musa genotype (n)

Roots

Medium

Pfb

Rrc

juveniles

females

males

total

juveniles

females

males

total

Pisang Jari Buaya (12)

2690 a
(250-4400)a

778 ab
(50-1325)

195 a
(50-375)

3675 a
(375-5975)

468 a
(183-1392)

191 a
(33-508)

72 a
(8-250)

732 a
(150-1941)

4454 a
(633-7800)

89

Gros Michel (12)

7005 b
(3875-14900)

938 abc
(425-1550)

261 ab
(175-475)

7128 b
(4750-16825)

1732 b
(533-3375)

726 b
(158-1783)

113 ab
(42-300)

2571 bc
(917-5025)

10902 b
(1442-21850)

218

Yangambi Km5 (12)

3943 a
(775-8025)

525 a
(200-825)

291 ab
(50-525)

5502 ab
(925-9275)

817 ab
(158-2117)

549 b
(33-1083)

52 a
(17-125)

1287 ab
(191-3150)

6998 ab
(1225-12375)

140

SH-3142 (12)

3178 a
(2150-3375)

637 ab
(400-925)

356 abc
(150-425)

4171 a
(2775-4675)

646 a
(142-1817)

294 ab
(67-400)

58 a
(8-158)

999 ab
(217-2375)

5171 a
(3867-5833)

103

Cachaco (12)

10762 b
(2375-17725)

1020 bc
(250-1875)

425 bc
(250-925)

13056 c
(2875-20525)

4254 c
(250-8383)

645 b
(8-1292)

271 b
(75-542)

5170 c
(258-9792)

18227 c
(3133-29500)

365

Grande Naine (12)

8763 b
(6575-11450)

1493 c
(900-2575)

493 c
(250-825)

10750 bc
(9000-13550)

2451 bc
(1642-3517)

614 b
(267-1475)

120 ab
(33-233)

3186 c
(2008-4900)

13936 bc
(17150-10708)

279

Data were log(x+1) transformed prior statistical analysis. Means in the same column followed by the same letter do not differ according the Tukey test (P £ 0.05).

a (minimum-maximum), b Pf = final population in roots and medium, c Rr = Pf / PI (with PI = 50 females)

It is important to note that there are indications that the resistance mechanisms of 'Pisang Jari Buaya', 'Yangambi Km5' and 'Gros Michel' are different [33-36]. Some mechanisms may be based upon intrinsic characteristics of the root tissue such as lignification ('Pisang Jari Buaya') or suberisation ('Yangambi Km5'), whereas others may be based upon physiological responses to nematode attack such as accumulation of phenolic compounds ('Gros Michel', 'Yangambi Km5') or differences in tannin levels. Overall, it seems that the resistance to R. similis in 'Pisang Jari Buaya' is a constitutive mechanical resistance, while the resistance in 'Gros Michel' and 'Yangambi Km5' is an induced post-infection resistance. In our in vitro screening experiments this second type of resistance does not appear to be expressed or switched on at 8 weeks after inoculation.

Experimental studies that compared resistance to R. similis of in vitro banana plants, corm-derived plants and field plants [18,37,38] suggest that the plant growth stage may influence resistance observations of some genotypes, for example FHIA-01 [28]. Especially in the early vegetative growth phase, probably not all types of resistance are expressed.

In vitro screening has several advantages compared to greenhouse screening. The experiments can be performed under controlled conditions (light, temperature, and medium). Furthermore, a small nematode inoculum is sufficient and can be prepared precisely by manually picking nematodes. Less space and time are needed for plant multiplication and maintenance. The experimental time is reduced to 8 weeks compared with screening in the greenhouse [8,19,23]; rooting of the plantlets is reduced from 8 to 4 weeks and the acclimatization period of 4 weeks is superfluous. The analysis (nematode extractions, nematode counting) is easier to handle since work is carried out on a smaller scale. A disadvantage, however, is the extraction of nematodes from the medium by maceration and sieving, as this is rather difficult and therefore time consuming. The agar can easily clog the sieves, causing very slow percolation of the suspension. Another important issue is the variable reproduction of the same nematode population between experiments. The reproduction ratio (Rr) of R. similis (Ugandan population) on in vitro 'Grande Naine' in the first, second and third experiment is very different. The age of the female nematodes and the overall quality of the nematode population can most likely explain these differences in reproduction. It is therefore very important to use a susceptible reference genotype, like 'Grande Naine' in all screening experiments. Finally, it is important to note that resistance picked up under in vitro conditions has to be confirmed under greenhouse conditions, and more importantly in the field.

5. CONCLUSION

Alfalfa callus on modified White's medium appeared to be a successful host to culture R. similis and P. coffeae under aseptic conditions. The system has many advantages: (a) the nematodes are free from bacteria and fungi; (b) subculturing is required less often than with carrot discs; (c) the nematodes are more easily available; and (d) the maintenance is labour intensive. The culturing system using alfalfa callus tissue did not affect the pathogenicity of the nematode populations compared with the same nematode populations cultured on carrot discs.

Both R. similis and P. coffeae were able to infect and reproduce on in vitro banana plantlets and in vitro A. thaliana. For A. thaliana, this opens the possibility of using this system as a model for rapid testing of interesting anti-nematode proteins, expressed in transgenic lines of A. thaliana (lectins, ribosome inactivating proteins, etc.). However, this model system needs further optimisation.

For Musa, an in vitro screening procedure has been successfully developed. It allows picking up resistance against the root-lesion nematode R. similis at a very early stage. In addition, this system could be useful to study resistance mechanisms. However, it is important to note that resistance picked up under in vitro conditions must be confirmed in a later phase under greenhouse conditions and finally in the field.

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[35] Pf, Final population of nematodes, including juveniles, females and males
[36] Rr, Reproduction ratio, final nematode population divided by the initial nematode population

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