THE BIOLOGY AND POSSIBLE IMPORTANCE OF APHELENCHOIDES

ARACHIDIS, A SEED-BORNE ENDOPARASITIC NEMATODE OF

GROUNDNUTS FROM NORTHERN NIGERIA

BY

JOHN BRIDGE 1), W. S. BOS 2), L. J. PAGE 1), and D. McDONALD 2)

1) Ministry of Overseas Development, Plant Nematology Laboratory, Imperial College of London
University, Ashurst Lodge, Sunninghill, Ascot, Berkshire, England, and 2) Crop Protection Dept.,
Institute for Agricultural Research, Ahmadu Bello University, Samaru, P.M.B. 1044, Zaria, Nigeria

Aphelenchoides arachidis Bos was found in northern Nigeria as a facultative endoparasite of

groundnuts (Arachis hypogaea L.). It occurs within the tissues of the pods, testas, roots and
hypocotyls, but not within the cotyledons, embryos or other parts of the plant. Many nematodes
within testas caused discolouration and were associated with shrivelled and small seeds. The
nematode is present in a small area around the town of Samaru, northern Nigeria. The nematodes
can affect the commercialvalue of confectionerygroundnuts, but have not been shown to diminish
yield. A simplified hot water treatment of seeds successfully controlled the nematodes, and other
methods of cultural control can be used.

In 1970, nematodes were found in seed testas of groundnut kernels examined

for fungal pathogens in the Crop Protection Department, Institute for Agricultural
Research, Samaru, Nigeria. These nematodes have been described as Aphelen-
choTde.r arachidis Bos (Bos, 1977). The nematodes were thought to constitute a
danger to the groundnut crop, and a research programme was started in Nigeria
and partly continued in England under glasshouse conditions. Observations and
studies were made on the general biology of the nematode, symptoms of damage
on a number of groundnut varieties, and its possible economic importance and
control.

MATERIALSAND METHODS
Biology of A. arachidis (md JytnptomJ of damage: All observations were made
on five confectionery groundnut varieties, S. 38, F. 439.4, US. 608, US. 617,
US. 628, and two other varieties, Spanish 205 and S. 61, all grown in northern
Nigeria.
Nematodes and eggs within the plant tissues were observed after staining in hot
0.1% cotton blue lactophenol and numbers of nematodes within each testa were
counted directly.
Live nematodes in infested testas and shells were extracted for counting by
shredding the tissues in shallow water in petri dishes and leaving for 72 hr, or
after blending whole pods for 5 sec in a household blender and leaving
overnight
on a cotton-wool filter in a shallow layer of water.
254

The behaviour of A. arachidis was observed on roots and fungi growing from
seeds of var. Spanish 205 in sterilised l jlo soil extract agar, and on fungi growing
from surface sterilised, detached testas on P.D.A. plates.
Nematode-free seeds of var. Spanish 205 were planted outside in concrete tanks
filled with infested soil, and fruiting pegs from the plants were labelled on, or
soon after, the day they reached the soil surface. Pods were harvested at different
times in 5-day periods, and the age of infected pods was determined.
Infested and nematode free seeds of var. Spanish 205 were dehydrated in an
alcohol series, embedded in paraffin wax (m.p. 54° C) and sectioned at 20 JLm
with a rotary microtome. Sections were stained in 0.1% methyl blue. Hypocotyl
tissue was stained in 0.1% cotton blue lactophenol and examined directly.
Hot water treätment: Infested seeds of var. F. 439.4 were first soaked in cold
water for 15 min and then immersed in five times their volume of water at 60°.
No further heat was applied. The seeds were left in the hot water for 5 min by
when the temperature had dropped to 54°. Fifty treated and 50 untreated seeds
were placed on moist tissue paper for 9 days and their germination compared.
Nematodes were counted from ten treated and ten untreated seeds.
In the glasshouse, hot water treated and untreated, infested seeds of var. S. 61
were planted in sterilised sandy loam in 23 cm plastic pots with ten replicates. The
groundnuts matured in 9 months. Seeds and pods were weighed and nematodes
counted in testas after staining whole seeds in o.I %o cotton blue lactophenol.

RESULTSAND OBSERVATIONS
S)lnzpton7.f: Seed coats of all varieties (Table I) were discoloured where there
were more than 2000 A. ?-?A?/testa, and more than 35,000 nematodes/testa
were often recorded. Some of the healthy looking seeds, varying from 4-30%,

TABLE I
E.rtinaated populations of Aphelenchoides arachidis and percentage infection of
discoloured compared to normal test,is of fite confectioner)' groundnut iarieties
grown in northern Nigeria
 255

were also infested with A. ?crachidis, but usually with fewer than 2000 nematodes/
testa. Heavily infested seeds, immediately after removal from fresh, mature pods,
had translucent testas, were a lighter brown and had darker vascular strands within
the testas. However, after the seeds had dried, infested testas were often wrinkled
and a darker brown. Nematodes were also found in relatively low numbers in
healthy seeds of other varieties.
Nematodes were found only in the tissues of the testa, mainly in the sub-
epidermal parenchymatous layer, and around the tracheids (Fig. 4a). Testas
infested with A. arachidis were thicker and more uneven than normal testas
(Fig. 4b). Nematodes were found in sub-epidermal parenchyma cells where walls
were broken and cells enlarged. The epidermal layer of the seed coat was reduced
in infested testas and the basal tissues including the aleurone layer were dis-
organised (Fig. 4a).
Many A. arachidÍJ were also found in pod shells of all groundnut varieties, and
also occurred in the roots and hypocotyl of var. Spanish 205, but not in any other
tissue of the plants. No necrosis or other symptoms could be attributed to the
presence of A. ärachidis in the roots or hypocotyl.
Infested seeds of var. Spanish 205 weighed less than healthy seeds, but nema-
tode damage had little effect on seed germination (Table II ) .

TABLE II

Comparison of .reed weights, ges°??ai?a?ttio?a,

and popttlations of A. arachidis from
normal and dis-colou?°ed .reed.r of groundnut var. Spanish 205

1) Means of ten replicates.

2) From 50 seeds.

Biology o f Aphelenchoides arachidis: Nematodes were found in the parenchym-

atous tissues of the testa (Fig. 1 ) , root cortex and hypocotyl, but not in the central
stele or vascular bundles. In the hypocotyl they occurred intercellularly (Fig. 2)
and most were orientated parallel to the central axis.
Many adult nematodes and eggs were found in the hypocotyls of emerging
volunteer plants of var. Spanish 205 growing in an infested field. Roots of the
same plants and 3-week old seedlings grown from infested seeds in sterilised soil
also contained A. arachidz.r.
Invasion of pods occurred 10 days after the fruiting pegs had penetrated the
soil. Few nematodes were found in pods up to 30 days, but thereafter nematode
numbers increased rapidly in infested pods (Table IV), with most found about day
60 with one pod containing over 5000 nematodes.
256

Development of A. arachidis occurred within the pod shells, testas, hypocotyl

and roots. In testas, all stages of the nematode were found throughout (Fig. 1),
but at the end of the growing season and in mature seeds, heavily infested testas
contained mainly juvenile stages with few adults. Testas showing no external
symptoms contained mostly adults and eggs, often arranged along the vascular
elments of the seed coats.
A. arachidis can survive desiccation in stored groundnut pods. However, only
2% and 3% of nematodes in testas of vars. U.S. 628 and F. 439.4 respectively
were alive after 4 months storage, although 5% were active in the testas of var.
Spanish 205 after 12 months (Table III). Most active nematodes were extracted
from testas of var. S. 61 immediately after harvest. More A. arachidis were found

TABLE III
Numbers of active and moribund A. arachidis extracted from testas, shells and
whole pods of different groundnut varieties

1) Means of ten replicates.

in shells than in testas, and more survived the same period of storage in shells.
Numbers of active nematodes varied from 14% to 31% of all those found in
shells for vars. F. 439.4 and U.S. 628 respectively. There were more than 66,000
active and moribund nematodes in shells of groundnut var. U.S. 628 and whole
pods of var. S. 61 immediately after harvesting contained 113,700 active nema-
todes. All juvenile stages were extracted live from dried testas and shells with no

TABLE IV
Numbers of A. arachidis found in pods of ground nut var. Spanish 205, sa?fa pled
. at different periods after penetration of the soil by pegs
 PLATEXXXVI

JOHN BRIDGE etal.: Aphelenchoides arachidis on groundnuts

Figs. 1. Surface view of nematode-infestedgroundnut testa (var. US. 628) showing juveniles and
eggs. of A. arachidi.rwithin the tissues. - 2. Eggs of A. arachidi.r found intercellularly within the
parenchymatoustissues of young seedlings of groundnut var. Spanish 205. - 3. Seeds of groundnut
var. F. 439.4 infested with nematodes (left) and healthy (right). - 4. Transverse sections of
groundnut testas (var. Spanish 205). a. infested with A. arachidi.r (N = nematodes); b. healthy
without nematodes.
 357

particular stage predominating, but only occasionally were adults found alive in
either testas or shells of stored pods. No active nematodes were extracted from
infested pods sun-dried in the field before storage.
Nematodes did not migrate out of the seed of var. Spanish 205 on agar plates
until germinating when many active juveniles moved through ruptures in the testa
caused by the emerging radicles. The nematodes moved freely in the agar and later
fed ectoparasitically on epidermal cells of the root and eventually invaded the
roots. They fed readily on mycelium of Macrophomina ph?t.seoli growing in the
agar from sclerotia on the seed surface, and fed and reproduced on Botryti.r
cinerea growing from surface sterilised testas of var. Spanish 205 on agar plates.

Hct waster treatnzent: All stages of A. arachidis were killed in seeds of var.
F. 439.4 treated with hot water, which treatment did not affect germination or
growth of seedlings (Table V).

TABLE V

Effect of bot water treatrnent on A. arachidis in seeds testas and germination of

groundnut IJar. F. 439.4

'
1) Means of ten replicates.
2) From 50 seeds.

There was no difference in yield of hot water treated and untreated seeds of
var. S. 61 grown in pots, and none of the pods produced from treated seeds were
infested with A. tlrachidiJ. 42 glo of seeds produced from untreated seeds contained
nematodes (Table VI).

TABLE VI

Effect of hot water treatment on percentage infestation with A. arachidis and yield
of grottndnut var. S. 61 grown in pots

1) Means of ten replicates.

DISCUSSION
Some seeds of all groundnut varieties examined from northern Nigeria were
infested with A. arachidi.r. This is the first record of a seed-borne nematode
infestation of groundnuts. Two other species of the genus are seed-borne in other
258

crops, namely A. bes.feyi, which causes white tip of rice (Cralley, 1952), and A.
ritzemabosi Oil Callistephui- chillenJis (Brown, 1956).
A. aracbidi.r is a facultative endoparasite of the seed testa, pod shell, roots and
hypocotyl of groundnuts. It also fed ectoparasitically on roots and on two fungi,
Macrophomina phaJeoli and Botrytis cinerea. The nematode probably feeds on
these fungi in soil in the absence of groundnuts although many other fungi
associated with groundnuts (McDonald, 1968a, b; 1969) may be a source of food
for A. al'achidis. Both fungi damage the groundnut crop; Macrophomina phaseoll
causes charcoal root and stem rot, and seedling, pod and seed rots, and Botrytis
cinerea causes a leaf and stem rot.
It was first thought that A. car?achidi.rinvaded pods by migrating through roots,
stems and pegs, but this was not confirmed: it seems more likely that nematodes
invade directly from the soil or infested seeds. In var. Spanish 205, the nematodes
increased in young pods from about four weeks after the pegs penetrated the soil
and pods had started to develop, but initial invasion also occurs in ten day old pods.
In infested pods, many nematodes migrate into the spaces between shell, testa and
cotyledons and are released when moisture is absorbed before germination and the
pods and testas burst. About this stage they can penetrate the young roots and
hypocotyls and reproduce in very young plants.
The nematode can survive desiccation in stored pods of groundnuts under certain
circumstances, but few remain alive after storage. All juvenile stages and some
adults can be revived from stored seeds, unlike other desiccation resistant nema-
todes that generally have a specialised survival stage in their life cycle, normally
the 2nd or 4th stage juveniles. Volunteer plants in an infested field contained
many adult nematodes suggesting that they continue to develop to maturity under
natural conditions in pods left in the ground during the dry season (October-
April) in Nigeria.
A. arachidis has not been found outside a limited area around Samaru, northern
Nigeria, apart from a single record of unidentified nematodes in groundnut testas
from Gwarzo village, near Kano, Nigeria: the distribution of this nematode in the
groundnut growing areas of northern Nigeria is now being surveyed. Since Samaru
is a major centre of introduction of new groundnut varieties to Nigeria, the pest
may have been imported.
These seed-borne nematodes devalue confectionery groundnuts because of the
associated shrivelled and discoloured seeds, but where the nematodes were found
confectionery varieties are unimportant.
Agricultural practice in northern Nigeria, especially sun-drying of the pods
after harvesting in very dry conditions (relative humidity can drop to 4%), con-
trols the nematodes under certain conditions. Shelling before planting eliminates
the tissues in which most nematodes occur and where they survive best. In southern
areas of Nigeria and other countries, where sun drying of pods is less efficient
because conditions are more humid, the nematodes may survive and be dispersed
in the seed and endanger the groundnut crop. Our observations
suggest that some
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varieties tested were less susceptible or relatively poor hosts for the nematodes, but
all varieties were infested to some extent.
A simplified hot water treatment (Bridge, 1975 ) is effective in killing all
nematodes within seed coats and requires only simple heating apparatus. Heavily
infested seeds are recognisable and could be sorted out before planting, but seeds
with few nematodes are indistinguishable from healthy seeds and, if planted, would
spread A. ctrctchidiJ. The hot water treatment may be useful to control the nema-
todes in groundnut growing areas with wetter conditions, if needed.

We thank K. Williamson and O. I. Kadir for technical assistance, and

J. M. Waller, Commonwealth Mycological Institute, and Mr. Audu, Institute for
Agricultural Research Samaru, for fungal identifications. Part of these studies
conducted in Nigeria and England was supported by the U. K. Ministry of
Overseas Development.

RÉSUMÉ
Biologie et importance possible d'Aphelenchoides arachidis, nématode endoparasite de l'arachide
transmis par les graines au Nigeria du Nord

Aphelenchoides arachidis Bos, 1977, observé au Nigeria du Nord, agit comme endoparasite de
l'arachide (Arachis hypogaea L.) ; il se localise en effet dans les tissus des coques, du tégument
séminal, des racines et des hypocotyles; il est absent par contre dans ceux des cotyledons, des
embryons et des autres parties de la plante. Les populations élevées du nématode présentes dans le
tégument séminal y causent des changements de coloration et sont associées à des graines petites
et rabougries. Au stade actuel des recherches, ce nématode apparait avoir une distribution restreinte
à une zone limitée autour de la ville de Samaru (Nigeria du Nord). Cette action du nématode peut
affecter la valeur des arachides de bouche, mais des réductions de rendement n'ont pas été établies.
Un traitement simple des graines à l'eau chaude a diminué l'incidence du nématode; d'autres
méthodes de lutte, culturales, pourraient être envisagées.

REFERENCES
Bos, W. S. (1977). Aphelenchoidesarachidis n. sp. (Nematoda: Aphelenchoidea), an endoparasite
of the testa of groundnuts in Nigeria. Z. Pfl. Krankh. Pfl. Schutz. (In Press)
BRIDGE,J. (1975). Hot water treatment to control plant parasitic nematodes of tropical crops.
Meded. Fac. Landbouw. Rijks. Univ. Gent, 1975, 40, 249-259.
BROWN,E. B. (1956). A seed-borne attack of chrysanthemum eelworm (Aphelenchoides ritzema-
bosi) on the annual aster (Callistephus chinensis). J. Helminth. 30, 145-148.
CRALLEY, E. M. (1952). Control of white tip rice. Arkans. Fm. Res., 1, 6.
McDONALD,D. (1968a). The effect of wetting dried groundnuts on fungal infection of kernels.
Samaru agric. Newsletter 10, 4-7.
- (1968b). A list of fungi associated with groundnuts in Nigeria. Samaru Miscellaneous Paper
no. 27. Ed. by Institute for Agric Res., Zaria, Nigeria.
- (1969). Groundnut pod diseases. Rev. appl. Mycol. 48, 465-474.