Evaluation of a Bioassay for Screening Resistance of Pineapple to Root-

Knot and Reniform Nematode

B.S. Sipes
Department of Plant and Environmental Protection Sciences
3190 Maile Way
University of Hawaii
Honolulu, HI 96822
USA

Keywords: Ananas comosus, bioassay, genetic modification, Meloidogyne javanica,

Rotylenchulus reniformis, tissue culture

Abstract
Accurate greenhouse bioassays of root-knot and reniform nematode
resistance in pineapple have required 9 to 12 months. We developed protocols to
evaluate pineapple for resistance to nematodes using a reproductive factor
(Rf=Pf/Pi) that shortened this time to 3 months for Meloidogyne javanica and to 6
months for Rotylenchulus reniformis. We used pineapple plants that had been
subjected to a genetic modification procedure using a cystatin gene. Plants were
transitioned from tissue culture to the greenhouse by repotting into individual pots
and allowing 1 month for growth before inoculation with nematodes. This protocol
was used for eight different sets of plants containing multiple pineapple lines. Tissue-
cultured plants of ‘Smooth Cayenne’ clone Champaka F-153 and hybrid D-10 that
had not been subjected to the transformation protocol were used as controls. The Rf
of M. javanica and R. reniformis on untransformed D-10 averaged 5.8 (s=10.28,
n=144) and 37.8 (s=59.19, n=64), respectively. On F153, the Rf was 32.9 (STD=37.17,
n=13) for R. reniformis. For those plants that had been subjected to a transformation
process, M. javanica Rf averaged 20.68 (s=35.57, n=389) and R. reniformis Rf
averaged 41.72 (s=70.88, n=332) over all of the pineapple lines. In Line 256, the Rf of
M. javanica and R. reniformis averaged 22.69 (s=21.12, n=79) and 28.47 (s=35.23,
n=97), respectively. In Line 101, M. javanica Rf was 4.7 (s=6.78, n=172). The
coefficient of variation associated with the Rf bioassays exceeded 100 in all cases
making conclusions difficult. The sets were composed of many lines of pineapple,
most represented by only a few plants. Plant size was not uniform at transplanting
and plant growth among lines was different. Differences arising from the time of
year the assay was conducted may also have contributed to the wide variation. The
transformation and genetic modification process appears to alter the host status of
pineapple, making some lines better hosts to the nematodes. Because of the
variation, it is necessary to produce and evaluate multiple plants in a line to
accurately identify nematode resistance. A rapid and reliable bioassay is critical for
evaluation of transgenic pineapple lines as well as very useful for evaluation of non-
transgenic pineapple plants.

INTRODUCTION
Plant-parasitic nematodes can severely limit pineapple yield (Sipes et al., 2005).
Fumigant nematicides were the primary control for nematodes until the 1980s after which
these pesticides were gradually removed from the market. One long term objective has
been to develop commercial pineapple cultivars with host-plant resistance to nematodes.
However, little resistance has been found among cultivated pineapple or its close relatives
(Collins and Hagan, 1932; Hagan and Collins, 1932; Sipes and Schmitt, 1994). If native
resistance is unavailable, a logical approach is incorporation of resistance via a
transgenetic approach. By the mid 1990s, the most successful transgenic approach to
nematode control had been through the use of a cystatin gene (Lilly et al., 1999) and a
pineapple transformation project was undertaken with one objective being to incorporate

Proc. 7th International Pineapple Symposium 185

Eds.: H. Abdullah et al.
Acta Hort. 902, ISHS 2011
nematode resistance (Rohrbach et al., 2000).
An accurate assay for nematode resistance is needed that is efficient in space,
time, and manpower. Previous assays employing crowns utilized pots 15 cm in diameter
or larger, and required 9 to 12 months for evaluations (Sipes and Schmitt, 1994). This
assay is not suited for pineapple that has been tissue cultured since the plants are small,
often less than 100 g. We conducted a nematode bioassay on putatively transformed
pineapple lines. The objective of this trial was to generate estimates of variance
associated pineapple putatively transformed for resistance to nematodes and estimate the
number of plants needed per line for an accurate assay for transgenic resistance.

MATERIALS AND METHODS

Ananas comosus, D-10 hybrid (Armstrong et al., 1979) and ‘Smooth Cayenne’
clone Champaka F-153, were used in the nematode assays. Putatively transgenic plants
were produced from these cultivars by particle bombardment of protocorm-like bodies
(Nan and Nagai, 1998) or by Agrobacterium-mediated transformation of leaf bases
(Wang et al., 2009). An engineered rice cystatin (Oc-IΔD86) and one of four promoters
(ubi9, Tubulin, 35S, ord CLCT- pineapple root specific) with either nptII or hpt as a
selection marker was used in the particle bombardment constructs. Agrobacterium
tumefaciens strain AGL0 carrying pCAMBIA with Oc-IΔD86, a CLCT or ubi9 promoter,
and an nptII selection marker gene. Plantlets that survived selection were maintained in
tissue culture until the shoots were 8-10 cm long. These plants were used in the nematode
bioassay.
Plants were removed from tissue culture in sets consisting of plants that had been
subjected to transformation at similar times or plants that had reached the desired size at
similar times. The results for eight sets of plants are reported on in this paper. Some sets,
contained wild-type D-10 or F153 plants that had only been subjected to tissue-culture
propagation. Each set contained different numbers of putatively transformed pineapple
lines that had survived on selective media with varying numbers of plants/line. For Line
256 and Line 101, initially positive in molecular assays for transformation, multiple
plants were propagated. The control D-10 plants used with Line 101 were subjected to the
transformation protocol but did not contain the cystatin gene.
Upon removal from tissue culture media, the plantlets were washed and weighed.
The clean plants were transplanted into 7.5-cm-d clay pots filled with a sterile soil/sand
mixture. The plants were placed on a greenhouse bench under 20% shade for 4 weeks to
minimize transplant shock. Plants were transferred to unshaded greenhouse benches and
arranged randomly.
The pineapple plants from sets 2, 3, 4, 5, 6, 7, and 8 were inoculated with 500 eggs
of Meloidogyne javanica delivered in 10 ml aliquots. Uninoculated controls received
10 ml of water. In lines with two to four plants, one plant was not inoculated with
nematodes to serve as a control. If a line was represented by five to six plants, then two
plants served as uninoculated controls. Plants were grown in the greenhouse for 3 months
and then the experiment was terminated. At harvest, roots were gently washed to remove
soil and then cut at the plant butt. Roots were shaken in NaOCl to extract eggs (Huessy
and Barker, 1973), dried at 70°C and weighed. Nematode eggs recovered from each plant
were counted and recorded.
The shoots from plants in sets 2, 3, 4, 5, 6, and 7 were weighed, cured for 1 week,
and replanted into 15 cm diameter pots filled with a sterile soil/sand mixture. Plants from
set 1 were removed from tissue culture media, washed, weighed and planted into a similar
sterile mixture. One month after replanting, the plants were inoculated with 1,000 eggs of
Rotylenchulus reniformis delivered in 10 ml aliquots. Uninoculated controls received
10 ml of water. The plants were grown for 6 months and then data were collected for root
weights and nematode populations as described for M. javanica.
Nematode reproduction (Rf) was calculated by dividing the eggs recovered from
each plant by the number of eggs inoculated onto the plant (Pf/Pi). Nematode eggs/g plant
root was also calculated. Sets were combined for a meta analysis of variance. Shoot

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weight and root dry weight were analyzed for variance. Variance was calculated for
nematode Rf, eggs/g root, shoot weight and root dry weight.

RESULTS
The plants generally grew normally upon removal from tissue culture. The
majority of lines originating from D-10 were of a normal phenotype. Only a few lines
exhibited abnormal phenotypes of either stunted growth or a narrow leaf. The F-153-
derived lines commonly had a spiny-leaf phenotype upon removal from tissue culture that
the plants did not grow out of. Inoculation with M. javanica or R. reniformis had
inconsistent effects on pineapple growth (Table 1). Plant growth was variable in
uninoculated controls as well as in inoculated transformed lines. Plant growth in the
untransformed D-10 and F-153 was also highly variable with the cultivars from 16 to over
63%.
The sets differed for Rf, (P <0.01) (Table 2). For M. javanica, the averages from
set 2 and 3 were consistently among the lowest values whereas averages for set 6 were the
highest. For R. reniformis, variation was greater among the sets compared to data for
M. javanica. This greater variation between sets was evidenced in the changes in ranking
from parameter to parameter.
Nematode Rf was not normally distributed in the transformed lines (Fig. 1) nor in
the control D-10 and F-153 plants (data not shown). Standard deviations were generally
high and exceeded the values for the mean for nematode associated measures as
compared to the plant growth parameters measured. The Rf of M. javanica and
R. reniformis on untransformed D-10 averaged 5.8 (s=10.3, n=144) and 37.8 (s=59.2,
n=64), respectively. On F153, nematode Rf was 131.8 (s=132.0, n=13) and 32.3 (s=37.2,
n=13) for M. javanica and R. reniformis, respectively. For plants subjected to
transformation, M. javanica Rf averaged 20.7 (s=35.6, n=389) and R. reniformis Rf
averaged 41.7 (s=70.9, n=332). Eggs/g dry root had similar coefficients of variance and
were correlated to Rf in M. javanica (r=0.13, P>0.001) but not in R. reniformis.
Line 256 plants weighed 26.9 g (s=15.1, n=79) and 20.5 g (s=13.3, n=13) without
nematodes at termination when inoculated with M. javanica. Similar growth was
observed with R. reniformis. Inoculated plants averaged 82.1 g (s=50.2, n=97) whereas
uninoculated plants averaged 58.8 g (s=33.9, n=17). Nematode Rf on Line 256 was 22.7
(s=21.1, n=79) for M. javanica and 28.5 (s=35.2, n=97) for R. reniformis.
In Line 101, M. javanica reduced final plant weight compared to the uninoculated
Line 101, 65.5 g (s=32.2, n=172) and 66.4 g (s=36.1, n=172) respectively. The D-10
controls averaged 105.6 g (s=47.59, n=115) without nematodes, whereas nematode
inoculation reduced D-10 growth to 104.6 g (s=52.04, n=115). Nematode Rf was 4.7
(s=6.8, n=172) on Line 101 and 3.4 on the D-10 control plants. (s=4.5, n=115). Line 101
was not challenged with R. reniformis.

DISCUSSION
The variance associated with nematode reproduction was large. This poses
challenges in biological assays of transgenic plants because sample sizes will have to be
quite large to detect differences in nematode resistance. Using data from all lines
combined and the formula

n = (t12s2)/d2 (1)

where t1 is the t value of the desired confidence interval, s2 is the variance, and d is the
half-width of the desired confidence interval (Steele and Torrie, p. 120), a sample size of
215 plants would need to be evaluated to detect a 90% reduction in M. javanica
reproduction with a 95% confidence level. A similar number of plants (210) would be
required to detect a similar reduction in R. reniformis reproduction. Using only Line 101
data, 150 plants would be required to detect a 90% reduction in M. javanica reproduction.
With Line 256 data, 69 plants would be sufficient to detect a 90% reduction in

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M. javanica reproduction. However, 121 plants would be needed for R. reniformis. These
sample sizes are substantially larger than what has been utilized in greenhouse
evaluations with crowns, slips or suckers (Collins and Hagan, 1932; Hagan and Collins,
1932; Sipes and Schmitt, 1994). These numbers, 69 to 215 plants, are reflective of
variation introduced by the transformation and tissue culture process. These high numbers
are prohibitive for assays of transgenic pineapple. It is therefore important to take steps to
reduce variability in the transgenic nematode assays.
Plant size, plant uniformity and environment have profound effects on nematode
reproduction. We observed these effects in these experiments, as larger plants had smaller
variance associated with plant growth and nematode reproduction. In soybean, the
evaluation of resistance for Heterodera glycines, the soybean cyst nematode, is affected
by plant vigor, methods used in the assay, and environmental conditions (Niblack et al.,
2002; Riggs and Schmitt, 1991). Transgenic pineapple may be no different. Niblack et al.
(2009) highlight the need for uniform plant size and vigor, uniform growing conditions,
the inclusion of stand susceptible checks, and use of a randomized complete block or
completely randomized experimental designs in the resistance evaluation for soybean cyst
nematode. In the experiments reported here, pineapple plants varied in size, arrived and
were assayed at different times of the year, and often susceptible checks were not
available for simultaneous evaluation.
Some variability may not be controllable. Micropropagation of pineapple can
introduce genetic variation into the plantlets (Feuser et al., 2003; Santos et al., 2008). We
observed this in the F153 plants as a spiny-leaf phenotype and in some D10 plants as
narrow leaves or stunted growth. The transformation protocol may also cause variation
that is not observed in vegetatively propagated field grown pineapple (Yabor et al., 2006).
As more efforts are directed towards development of transgenic nematode
resistance in pineapple, it will be important to employ an assay that minimizes variance.
Lines surviving selection should be positively transformed, i.e., Southern DNA assay
positive and have sufficient expression levels (if that is the mechanism of resistance).
These select lines will need to be multiplied to provide replicates that are vigorous and
uniform for the bioassay. Susceptible checks will also need to be multiplied alongside the
transformed lines to provide appropriate comparative controls. Appropriate check plants
might include (1) a standard susceptible cultivar (i.e., F-153), (2) the desired cultivar (i.e.,
‘73-50’), and (3) the desired cultivar transformed with a marker gene (i.e., ‘73-50’ with
GFP). All of the check plants should be tissue-cultured and of similar size and vigor as
the transformed lines being tested.
ACKNOWLEDGEMENTS
Drs. C. Nagai, M.-L. Wang, P. Moore, and staff at the Hawaii Agriculture
Research Center developed the plants. Dr. R. Paull and G. Uuru at the University of
Hawaii contributed to the development of the transgenic plants also. Dr. H. Atkinson with
the University of Leeds allowed the use of the modified cystatin construct and provided
valuable discussion on analysis of efficacy. D. Meyer, G. Nagai and M. Young
contributed nematological expertise in the experiment. This work was supported in part
by special grants from the USDA and Hatch funding administered by the College of
Tropical Agriculture and Human Resources, University of Hawaii.

Literature Cited
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of Heredity 23:459-465, 503-511.
Feuser, S., Meler, K., Daquinta, M. and Guerra, M.P. 2003. Genotypic fidelity of
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Rohrbach, K.G., Christopher, D., Hu, J., Paull, R., Sipes, B., Nagai, C., Moore, P.,
McPherson, M., Atkinson, H., Levesley, A., Oda, C., Fleisch, H. and McLean, M.
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Santos, M.D., Buso, G.C. and Torres, A.C. 2008. Evaluation of genetic variability in
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Tables

Table 1. Shoot weights of pineapple subjected to genetic transformation and inoculated

with nematodes. D-10 is a hybrid of unknown parentage (Armstrong et al., 1979 and
Champaka F-153 is a ‘Smooth Cayenne’ clone.

Meloidogyne javanica Rotylenchulus reniformis

Shoot wt. (g) s1 n Shoot wt. (g) s n
Uninoculated lines 56.5 35.1 262 74.1 49.2 94
Inoculated lines 54.4 31.6 389 108.1 61.5 332
Uninoculated D10 100.0 49.7 125 111.7 71.1 64
Inoculated D10 93.1 53.3 144 77.9 52.0 15
Uninoculated F153 48.0 7.6 12 107.6 28.0 13
Inoculated F153 49.2 17.0 13 100.2 23.9 12
1
n = number of observations and s = standard deviation.

Table 2. The shoot weight and nematode reproduction (initial nematode population
inoculated onto the plant/the nematode population recorded on the plant at harvest) for
sets of transgenic pineapple assayed.

Shoot weight (g) Rf

Set n1
X s CV X s CV
Meloidogyne javanica
2 41 39.39 18.49 47 4.55 5.15 113
3 17 25.75 11.69 45 2.16 2.47 114
4 35 34.54 15.76 46 8.57 7.21 84
5 98 31.82 18.86 59 20.40 20.34 99
6 61 55.71 35.43 64 54.27 62.68 115
7 81 49.05 24.50 50 24.49 34.80 142
8 172 65.50 32.19 49 4.73 6.78 143
Rotylenchulus reniformis
1 34 99.86 76.91 77 22.71 13.34 59
2 40 128.32 52.96 41 26.85 52.25 195
3 17 103.43 40.61 39 20.54 19.85 97
4 35 167.16 59.74 36 44.56 64.41 145
5 99 86.12 58.62 68 54.07 95.82 177
6 61 106.61 67.75 64 64.31 72.06 112
7 83 66.50 34.22 51 15.97 31.25 196
1
n = number of observations, X = average, s = standard deviation and CV = coefficient of variation.

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Figures

Fig. 1. Distribution of nematode Rf (reproductive factor = Pf/Pi) in pineapple, Ananas

comosus, subjected to transformation with a modified cycstatin gene and
challenged with Meloidogyne javanica or Rotylenchulus reniformis.

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