Major Diseases of Groundnut PDF

M a j o r Diseases of Groundnut
Compiled by
Faujdar Singh and D.L. Oswalt
Skill Development Series no. 6
ICRISAT
Human Resource Development Program
I n t e r n a t i o n a l C r o p s Research I n s t i t u t e f o r the S e m i - A r i d T r o p i c s
P a t a n c h e r u , A n d h r a Pradesh 502 324, I n d i a
1992
Major Diseases of Groundnut
Compiled by
Faujdar Singh and D.L. Oswalt
Skill Development Series no. 6
ICRISAT

I n t e r n a t i o n a l C r o p s R e s e a r c h I n s t i t u t e for t h e S e m i - A r i d T r o p i c s
P a t a n c h e r u , A n d h r a P r a d e s h 502 3 2 4 , India
1992
Publications in this Skill Development Series (SDS) are issued

semiformally for limited distribution to program p a r t i c i p a n t s ,
colleagues, and collaborators. Copies may be requested by the SDS number.
Constructive criticism from readers is welcomed by: Program Leader, Human
Resource Development Program, ICRISAT.
Acknowledgments
Information has been taken from published and unpublished reports. This
publication should not be cited as a reference.
C o m m e n t s , s u g g e s t i o n s , and e n c o u r a g e m e n t f r o m D r s D.V.R. R e d d y , D.H

S m i t h , P . S u b r a h m a n y a m , S.B S h a r m a , N . R a m a K r i s h n a , D . M c D o n a l d , a n d
Y.L. Nene for compiling this document are gratefully acknowledged.
T h a n k s t o M r S.V. P r a s a d R a o for c o m p u t e r i z i n g t h i s m a n u s c r i p t .
Contents
Introduction 5
Fungi 5
Bacteria 6
Viruses 6
Nematodes 6
Disease Assessment 7
Diseases o f Groundnut 8
Fungal Diseases 8
Bacterial Disease 8
Virus Diseases 8
Nematode Diseases 8
MP 1. Seed Rots and Seedling Diseases of Groundnut 9

MP 2. Groundnut Pod Rots 10
MP 3. Groundnut Yellow Mold 10
MP 4. Determination of Seed Resistance to Colonization by
Aspergillus flavus 11
MP 5. Groundnut Rust 12
MP 6. Rust Screening in the Field 13
MP 7. Rust Inoculation in the Laboratory 15
MP 8. Early and Late Leaf Spots of Groundnut 16
MP 9. S c r e e n i n g for E a r l y and L a t e Leaf Spots 17

MP 10. Use of Cob's Diagram for Percentage Leaf Area Damage
by Rust and Early and Late Leaf Spots 21
MP 11. Bud Necrosis 22
MP 12. Groundnut Rosette 23
MP 13. Peanut Clump 24

MP 14. Detection and Inoculation of Plant Viruses 24
MP 15. Bacterial Wilt of Groundnut 29
MP 16. Methods to Identify Plant Parasitic Nematodes 30
MP 17. Root-Knot Nematode 32

MP 18. Root Lesion Nematode 33
MP 19. Ring Nematode 34
MP 20. Testa Nematode 34
MP 21. Sting Nematode 35
References 36
Annexure I 38
Annexure II 39
Evaluation 40
Introduction
Disease is an alteration in one or more normal physiological processes,
resulting in a loss in utilization of energy in p l a n t s . The concept of
disease embraces any loss of a plant's ability to function normally or to
coordinate the production and utilization of energy. Organisms causing
diseases are fungi, bacteria, v i r u s e s , and n e m a t o d e s .
Fungi
Fungi are nonchlorophyllous, nucleated, unicellular, or multicellular

filamentous bodies that are reproduced by sexual or asexual spores.
Plant pathogenic fungi survive in soil, seed, and w e e d s . These are
dispersed by insects, wind, water, and animals. Fungi live as either
saprophytes on dead tissue or as parasites on living tissue. Some fungi
are biotrophs, i.e., active only in the living host. The fungal
pathogens are classified into two major divisions, Myxomycota and
Eumycota (Ainsworth et al. 1 9 7 3 ) .
Myxomycota. This group includes unicellular fungi that produce

Plasmodium or pseudo Plasmodium. These fungi are mostly propagated
by motile zoospores formed in Plasmodium. Resting spores are also
produced.
Kumycota. This group consists of filamentous fungi. These are

classified into five subdivisions based on sexual spores.
Mastigomycotina. They produce asexual spores in sporangia. They

include downy mildews and many pathogenic species of the water mold
genera. Their mycelium is mostly nonseptate, and the spores
liberated from the sporangia may be motile in some cases. Many
species cause seed decay and seedling failure of plants that are
grown in w e t s o i l s . T h e e x a m p l e s a r e Pythium a n d Phytophthora.
Zygomycotina. T h e s e i n c l u d e Rhizopus spp. and Mucor spp. The

sexual spores are zygospores.
Asconycotina. These fungi destroy foliage and parts of plants that

are above ground by abundantly producing ascospores on infected
foliage that spreads by wind and w a t e r . Filamentous ascomycotina
produce several spores that spread with splashing rain or by air.
It includes powdery m i l d e w and sexual stages of fungi such as
Aspergillus flavus, Cerospora arachidicola, and Cercosporidium
personatum.
Basidioaycotina. These include rusts and smuts that are host

specific p a t h o g e n s , and often have a complex life cycle.
Deuteromycotina, or Fungi imperfecti. In these fungi the sexual

reproductive stage is unknown or seldom found. This group
i n c l u d e s l e a f - s p o t t i n g f u n g i s u c h a s s p e c i e s o f Alternaria,
Cercospora, Phoma, and Colletotrichum. The perfect stage, i.e.,
teleomorph, if found, are usually in ascomycotina, although some
are in Basidiomycotina.
HRDP SDS no. 6 5

Bacteria
Bacteria are unicellular, primitive, plant-like, procaryotic organisms,
and lack an organized nucleus. Plant pathogenic bacteria are mostly rod
shaped, nonmotile, or motile by means of one or more flagella on their
body. Plant pathogenic bacteria are usually gram-negative. The bacteria
are mainly dispersed through seed, soil, air, and water. Sometimes
insects also transmit bacterial cells. Bacterial infection in plant
causes specific diseases such as blight, soft rot, leaf s p o t s , t u m o r s ,
cankers, and vascular w i l t .
Viruses
The word 'virus' means poison or the poisonous element by which infection
is communicated. The virus can be defined as a transmissible parasite
w h o s e n u c l e i c acid g e n o m e is less than 3 x 108 d a l t o n s in m a s s and t h a t
need ribosomes and other components of their host cells for
multiplication (Gibbs and Harrison 1 9 7 6 ) . Plant viruses are
submicroscopic entities showing obligate relationship with living cells
of the host and ability to cause specific diseases. The majority of the
plant viruses possess the following characteristics (Feakin 19 7 3 ) .
o They are composed of protein and nucleic acid.
o They contain one type of nucleic acid, either RNA, or DNA.
o They multiply only in the host cell (obligate pathogens).
o The RNA and protein subunits are formed separately in the host
cell and combine to form intact virus particles.
o Viruses do not possess enzyme systems, required to perform

metabolic processes.
o They are transmitted by graft inoculations, by sap, by

insects, and by nematodes.
An agent t h a t transmits a virus is called a vector. In many

plants, viruses are transmitted under natural condition by insects. Some
of them are also transmitted by mechanical sap inoculation. Viruses are
also transmitted by seed, e s p e c i a l l y i n t h e p l a n t s o f Leguminosae.
Nematodes
Nematodes (Greek for t h r e a d ) are e l o n g a t e , tubular organisms that m o v e

like a snake. These are nonsegmented round worms, sometimes referred to
as eelworms or simply nemas. Their body is long, narrow, and the
internal organs consists of a set of tubes enclosed by the body w a l l .
The alimentary canal runs directly back from the anterior mouth chamber
(stoma) to the posterior anus. The excretory system is a long coiled
tube or set of tubes that discharge to the exterior through a duct in the
body wall in the anterior third of the body. There are no blood vessels.
The nervous system consists of a limited number of nerve cells clustered
in a group anteriorly and another posteriorly. Nerve fibers extend along
6 HRDP SDS no. 6

the body wall. Most, n e m a t o d e s a r e c i r c u l a r i n c r o s s s e c t i o n . The dorsal
(back) region differs from the ventral. The right and left lateral
sectors are d i s t i n c t from either the dorsal or v e n t r a l sector (Fig. 1 ) .
The sexes are separate in most nematode species. Females are larger
than m a l e s . The female gonad consists of one or two elongated tubes.
The gonad w a l l is a single layer of flat cells forming a tube with many
distinct regions. The ovary is at the distal end. The region of cell
division (germinal zone) contains small cells, called oocytes. These
e n l a r g e and in t h e growth zone a c c u m u l a t e t h e c e l l u l a r m a c h i n e r y for
embryo formation. The gonad lining changes and the gonad narrows in the
next section (oviduct). This connects the ovary to the uterus, a region
of enlarged diameter. A pouch-like structure, the spermatheca is
situated between the oviduct and uterus. The sperms are stored in
spermatheca. From the u t e r u s , a short muscular vagina leads to a ventral
opening through the body w a l l , the vulva (Dropkin 1 9 8 0 ) . In some species
males have lateral cuticular extensions in the tail region, the caudal
alae, also called the bursae (Dropkin 1 9 8 0 ) .
Disease Assessment
To protect a crop from diseases, it is important to know the causal

o r g a n i s m s , symptoms, method of infection, and the methods for recording
observations and for correct interpretation of d a t a . The quantitative
study of damage to plants due to disease is called pathometry or
phytopathometry. The pathometric methods are visual assessment, severity
relationship ( I - S ) , inoculum disease relationships ( I - D ) , remote sensing
or video image analysis (Mayee and Datar 1 9 8 6 ) . The former three are
frequently used for groundnut disease assessment.
Figure 1. A plant parasitic nematode. (Source: Dropkin 1980)
HRDP SDS no. 6 7

Diseases of Groundnut
Fungal Diseases
In groundnut, fungi cause seed rots and seedling diseases such as root
r o t , s t e m r o t , w i l t s , b l i g h t , .pod r o t ; a n d f o l i a r d i s e a s e s s u c h a s r u s t
and early and late leaf spots.
Seed rots and seedling diseases. Many soil inhabiting fungi infect
a n d d a m a g e t h e s e e d a n d g e r m i n a t i n g s e e d l i n g s o f g r o u n d n u t (MP 1
and MP 2 ) . They may be identified by fungal spores that give
characteristic colorations to the seed, e.g., gray spores indicate
Rhizopus arrhizus, b l a c k s p o r e s a r e A s p e r g i l l u s niger, and green or
blue spores are Panicillium sp. T h e f u n g u s , A s p e r g i l l u s flavus,
p r o d u c e s a f l a t o x i n a n d c a u s e s a f l a r o o t of g r o u n d n u t (MP 3 a n d MP
4).
Foliar diseases. The major foliar diseases of groundnut caused by

f u n g i a r e r u s t (Puccinia arachidia S p e g . ) (MP's 5, 6, and 7 ) , late
leaf spot (Cercosporidium personatum r e c e n t l y r e n a m e d
P h a e o i s a r i o p s i s peraonata B e r k & C u r t ) , a n d e a r l y l e a f s p o t
(Cercospora arachidicola H o r i ) ( M P ' s 8 , 9 , a n d 1 0 ) . Rust and late
leaf spot are important diseases in India and most of the
semi-arid tropic (SAT) regions. Early leaf spot is an important
disease in Africa, particularly in southern Africa.
Bacterial Disease
Bacterial wilt of groundnut caused by Pseudomonas solanacearum E.F. Sm.

was first reported from Indonesia (1905) and later in Georgia, USA
(1931). Presently, this disease is distributed worldwide in cultivated
a r e a s o f g r o u n d n u t i n c l u d i n g A s i a a n d A f r i c a (MP 1 5 ) .
Virus Diseases
The major virus diseases of groundnut are bud necrosis, clump, rosette,
p e a n u t s t r i p e , a n d p e a n u t m o t t l e (MP 1 1 - 1 4 ) . Peanut clump virus is
t r a n s m i t t e d b y t h e f u n g u s Polymyxa g r a m i n i s ; c h l o r o t i c a n d g r e e n r o s e t t e
viruses are transmitted by aphids; bud necrosis virus causing bud
necrosis disease is transmitted by thrips. Peanut mottle and peanut
stripe viruses are transmitted by aphids.
Nematode Diseases
Several nematodes are parasitic to groundnut. These are root-knot

nematodes (Meloidogyne arenaria, Meloidogyne hapla, Meloidogyne
javanica), root lesion nematode (Pratylenchus brachyurus), ring nematode
(Macroposthonia ornata), sting nematode (Belonolaimus longicaudatus), and
testa nematode (Aphelenchoides arachidis) in MP's 16—21.
8 HRDP SDS no. 6

MP 1. Seed Rots and Seedling Diseases of
Groundnut
The important fungi-causing seed rots and seedling d i s e a s e s , and their
symptoms are given in Table 1 (Subrahmanyam and Ravindranath 1988).
T a b l e 1. P u n g i c a u s i n g seed rots and seedling d i s e a s e s of g r o u n d n u t .
Fungi Disease Symptoms
Aspergillus flavus Aflaroot or Affected seeds are shriveled and dried,

y e l l o w mold covered b y y e l l o w o r g r e e n i s h s p o r e s .
C o t y l e d o n s s h o w n e c r o t i c lesions w i t h
reddish brown margins. Seedlings are
h i g h l y s t u n t e d , leaf s i z e g r e a t l y r e d u c e d ,
w i t h p a l e t o light g r e e n c o l o r .
Aspergillus niger Crown rot or Germinating seeds are covered with masses
c o l l a r rot o f b l a c k c o n i d i a , rapid drying of p l a n t s .
L a t e r , w h o l e c o l l a r region b e c o m e s shaded
and d a r k b r o w n .
Fusarium solani and Wilt L o w e r end o f t a p r o o t b e c o m e s b r o w n t o

Fusarium oxysporum reddish b r o w n . Secondary roots become
b r o w n and b r i t t l e . Leaves turn grayish
green and p l a n t s d r y .
Rhizopus arrhizus and Seed and Sudden w i l t i n g of lateral b r a n c h e s that

sclerotium rolfsii seedling r o t , are completely or partially in contact
s t e m rot with soil. White coating of fungus
mycelium on affected plants.
Rhizoctonia solani Root rot, pod P r e e m e r g e n c e death of s e e d l i n g s ;

break down, shrunken, elongate dark brown areas on
wilt o n the h y p o c o t y l . T h e decayed a r e a s a r e
covered w i t h light-brown m y c e l i u m .
Pythium ultimum and Damping off Soft rot of the h y p o c o t y l r e g i o n c a u s i n g

Pythium myriotylum damping o f f .
Verticillum alboatrum Vascular wilt W i l t i n g o f leaflets a n d p e t i o l e s ,

leaflets a r e curled a n d c h l o r o t i c .
Control Measures
o Follow a crop rotation, i.e., cereal-cereal-groundnut.
o Sow good quality and disease-free seed.
o Avoid damage to the seed testa and deep placement of seed at

sowing.
o Treat the seed with thiram e 3 g kg-1 seeds or with carbendazim % 2

-1
g kg seeds.
HRDP SDS no. 6 9

MP 2. Groundnut Pod Rots
Most of the pod rots are due to the combined attack of several fungi.
I m p o r t a n t p a t h o g e n s o f t h e p o d r o t c o m p l e x a r e Fusarium solani, F.
oxysporum, Macrophomina phaseolina, Rhizoctonia solani, Sclerotium
rolfsii, and Aspergillus niger.
The occurrence of pod rots may be severe due to attacks of

nematodes, termites, and millipedes on pods, thus predisposing the
invasion by fungi and bacteria (Reddy and McDonald 1 9 8 3 ) .
Control Measures
o Use cereal-cereal-groundnut crop rotation and seed treatment with
thiram.
o Avoid drought at pod formation and maturity.
o Avoid heavy irrigation before harvesting. It will lead to pod rot

if harvesting is delayed.
o Grow varieties tolerant to stem and pod rots : ICGV 87157, or ICGV
86590.
MP 3. Groundnut Yellow Mold

T h e y e l l o w m o l d f u n g u s , Aspergillus flavus, is commonly found in the seed
of both rotten and apparently healthy pods of groundnut. Many strains of
this fungus are capable of producing aflatoxins that render the seed
unacceptable due to high toxicity for human or animal consumption (Reddy
and McDonald 1 9 8 3 ) . Aflatoxin contamination in groundnut can occur in
the stems of seedlings, pods, and seeds. The fungus is capable of
invading groundnut seeds before harvest, during postharvest drying, and
during storage.
T h e A . flavus g r o u p s o f f u n g i a r e f a c u l t a t i v e p a r a s i t e s . They
invade plant tissues directly or attack tissues that have been
predisposed by environmental stresses such as dry weather or damages
caused by insects, nematodes, natural cracking, and harvest equipment
(Pettit 1 9 8 4 ) .
Aflatoxins a r e c a r c i n o g e n i c a n d p r o d u c e d b y t h e Aspergillus flavus

g r o u p o f f u n g i t h a t h a v e b e e n identified a s B 1 , B2, G 1 , a n d G2. T h e
maximum aflatoxin level for groundnut acceptable in U S A is 20 p p b (Pettit
1984).
Symptoms
Yellow mold first appears on groundnut cotyledons after the emergence of

seedlings. Necrotic spots become covered with masses of yellow-green
s p o r e h e a d s o f t h e A . flavus g r o u p o f f u n g i . Fungus toxins are
10 HRDP SDS no. 6

translocated throughout the seedling in the transpiration stream.
Infected plants generally become stunted with symptoms of vein clearing
chlorosis on the leaflets. Such seedlings lack a secondary root system,
a c o n d i t i o n k n o w n as "aflaroot." Y e l l o w - g r e e n Aspergillus c o l o n i e s
develop on overmature and damaged seeds and p o d s .
Control Measures
o Harvest at proper maturity and discard the wilted and dead plants
as such plants are likely to have seeds infected b y Aspergillus
flavus.
o Dry the groundnut pods to 6-8% moisture content immediately after
harvesting and discard the infected pods and seeds.
o Avoid damage to the testa while decorticating.
o Prevent drought stress, and also prevent water logging (40-80% of

field capacity) at late stages of growth.
o Control nematode because they may predispose plants to attack by
fungal pathogens.
MP 4. Determination of Seed Resistance to

Colonization by Aspergillus flavus
To determine the invasion and colonization by yellow mold (Aspergillus
flavus) i n g r o u n d n u t , M e h a n a n d M c D o n a l d ( 1 9 8 3 ) suggested the following
procedure:
1. Take nondamaged, mature pods from each plot to provide:

a. 50 g seeds for m o i s t u r e d e t e r m i n a t i o n , and
b. 20 g seeds for t h e c o l o n i z a t i o n t e s t .
2. Use sample 'a' for m o i s t u r e d e t e r m i n a t i o n .
3. P l a c e 20 g s e e d s (sample 'b') in a c l e a n b e a k e r w i t h s u f f i c i e n t
aqueous solution of sodium hypochlorite (0.5%) to rover the seeds. Soak
seeds for 2 m i n , drain off excess solution, then rinse seeds in two
changes of distilled water. Drain-off water.
4. Hydrate seeds to about 2 0 % moisture content by soaking them for 10-15

min in distilled water. The exact time of soaking is determined by the
initial moisture content of the seeds.
5. Place seeds aseptically in a sterile petri dish (9 cm diameter) and

a p p l y 1 m L o f a s u s p e n s i o n o f s p o r e s o f a t o x i g e n i c s t r a i n o f A . flavus
(4 x 10' conidia mL-1) in distilled w a t e r . Now roll the seeds gently
around the dish to spread the inoculum evenly over their surfaces. The
s p o r e s u s p e n s i o n s h o u l d b e p r e p a r e d f r o m 8-10 d a y - o l d c u l t u r e .
HRDP SDS no. 6 11

6. The petri dishes are arranged over water in semirigid plastic boxes
provided with close fitting lids. Seal the lids with cellotape and place
t h e b o x e s i n a n i n c u b a t o r a t 25°C f o r 8 d a y s .
7. Remove boxes and petri dishes after 8 days of incubation. Examine

seeds under a stereoscopic-microscope for infection and colonization by
A . flavua. This should be d o n e with the m a t e r i a l in a laminar flow hood
or in a chamber with the air voided to the outside. It is necessary to
wear a surgical mask and gloves while recording the data.
8. The observations recorded are:

a. N u m b e r of s e e d s p e t r i d i s h - 1 (A)
b. Number of seeds with sporulating growths of A. flavus on their
s u r f a c e s (B)
c. Percentage of seeds invaded = B x 100
A
9. To classify the level of resistance to invasion and colonization by
A. flavus, use the following criteria:
a. Resistant = Sporulating growth on less than 15% of the seeds,
with growth and sporulation sparse.
b. Moderately resistant = Sporulating growth on 16-30% of seeds,
sporulation moderate to dense.
c. Susceptible = Sporulating growth on 31-50% of seeds,
sporulation dense.
d. Highly susceptible = Sporulating growth on over 5 0 % of seeds
with dense growth and sporulation.
MP 5. Groundnut Rust
Symptoms
Rust (Puccinia arachidis) i s i d e n t i f i e d b y t h e a p p e a r a n c e o f o r a n g e

pustules (uredinia) on the abaxial (lower) surface of leaves and reddish-
brown urediniospores (uredospores). Symptoms are mainly confined to
leaflets but pustules can be seen on all the aerial parts of a plant
except the flower. Brown to dark reddish-brown pustules appear on the
lower surface with the upper surface developing yellow, chlorotic spots
with necrotic brown areas in the center. At a late stage, the primary
pustules are surrounded by secondary sori. The uredinias are usually
c i r c u l a r , 0.3 m m — 1.0 m m i n d i a m e t e r , a n d c a n d e v e l o p o n a l l t h e a e r i a l
parts of the plant except flowers and pegs.
Control Measures
o A c e r e a l - c e r e a l - g r o u n d n u t c r o p r o t a t i o n and r e m o v a l of v o l u n t e e r
groundnut plants from the field will help to check rust inoculum
build-up.
o Adjust the sowing time to avoid the most conducive environmental

condition for rust development (i.e., high humidity, cloudy
weather) to help reduce damage caused by rust.
12 HRDP SDS no. 6

o Sprays of bordeaux mixture and dithiocarbamate have been found
effective to control rust and late leaf s p o t s . Chlorothalonil 0.2%
spray has been found effective against rust and late leaf spot,
when sprayed 30 days after germination till 15 days before
harvesting at regular 10-15 day intervals. However, this schedule
could be modified using a suitable disease forecast system based on
temperature, humidity, cloudy weather, and rainfall pattern to save
the fungicide and reduce the spray cost. Calixin is effective
against rust but not against leaf spots, whereas benomyl is
e f f e c t i v e a g a i n s t leaf spots b u t not a g a i n s t r u s t (Subrahmanyam et
al. 1984).
o Grow resistant cultivars: ICGV 87160 or ICGV 86590.
MP 6. Rust Screening in the Field

The screening of genotypes to assess rust resistance in the field is done
at two stages (Subrahmanyam et al. 1 9 8 0 ) .
Preliminary screening. Preliminary screening is carried out in

n o n r e p l i c a t e d p l o t s ( 1 r o w , s p a c e d 6 0 - c m a p a r t a n d 4-m l o n g ) a t I C R I S A T
Center. The highly susceptible standard checks are grown as infector
rows throughout the field with every one to four rows of the test
genotype. E a c h t e s t g e n o t y p e i s s c o r e d u s i n g a 1-9 s c a l e o n e w e e k b e f o r e
harvesting. Genotypes scored 1 to 5 are selected for advanced screening
and genotypes with scores between 6 to 9 are discarded. The description
of the disease scale is in Table 2.
Table 2. D e s c r i p t i o n o f groundnut rust scoring s c a l e ( 1 t o 9 ) .
Score Description D i s e a s e s e v e r i t y (%)
1 No disease. 0
2 L e s i o n s sparsely distributed largely at lower l e a v e s . 1- 5
3 M a n y lesions on lower l e a v e s , n e c r o s i s e v i d e n t ; v e r y few lesions

on middle leaves. 6- 10
4 N u m e r o u s lesions p r e s e n t o n lower and m i d d l e l e a v e s ; severe

n e c r o s i s on lower l e a v e s . 1 1 - 20
5 S e v e r e n e c r o s i s o f lower and m i d d l e l e a v e s ; lesions m a y b e

on t o p leaves b u t less s e v e r e . 2 1 - 30
6 E x t e n s i v e d a m a g e t o lower l e a v e s . Lesions densely present on

m i d d l e leaves with n e c r o s i s ; lesions a l s o o n top l e a v e s . 3 1 - 40
7 S e v e r e d a m a g e t o lower and m i d d l e l e a v e s ; lesions d e n s e l y

d i s t r i b u t e d o n top l e a v e s . 4 1 - 60
8 1 0 0 % d a m a g e t o lower and m i d d l e l e a v e s ; lesions o n top

leaves w i t h severe n e c r o s i s . 6 1 - 80
9 A l m o s t a l l leaves w i t h e r i n g ; b a r e stems p r e s e n t . 81-100
HRDP SDS n o . 6 13
Advanced screening. Advance screening is done by growing genotypes in
r e p l i c a t e d p l o t s (at l e a s t t h r e e r e p l i c a t i o n s ) o f t h e s a m e p l o t s i z e a s
in preliminary screening plots. Each test plot is separated by an
infector row that is a mixture of susceptible genotypes. The infector
rows are sown 2 weeks before the test material. Infector rows are
inoculated with a urediniospore suspension at flowering, using the
artificially inoculated potted 'spreader' plants. Such potted plants are
placed throughout the field to serve as an additional source of inoculum.
A f t e r i n o c u l a t i o n , t h e f i e l d i s i r r i g a t e d w i t h a purfo i r r i g a t i o n s y s t e m ,
on alternate days or as required until harvest. The observations are
r e c o r d e d u s i n g t h e 1—9 s c a l e ( F i g . 2 ) .
1 2 3
4 5 6
7 8 9
Figure 2. D i a g r a m s o f r u s t d a m a g e s (1—9 s c a l e ) o n g r o u n d n u t p l a n t s .
(Source: P. Subrahmanyam, ICRISAT, personal communication 1990)
14 HRDP SDS n o . 6
MP 7. Rust Inoculation in the Laboratory
Rust inoculation in the laboratory is done using the following
procedures:
1. Rust spores from infected leaves are collected in a glass tube with
the help of a 'cyclone spore collector'.
2. The collected spores are suspended into distilled water with a few
drops (1 mL lot-1) of w e t t i n g a g e n t (Tween 8 0 ) . Complete
suspension is made either by magnetic stirring or by manual
shaking.
3. Inoculum is adjusted to approximately 50 000 spores mL-1 of

solution. The spores are counted with the help of a
'Hemocytometer'.
4. The inoculum is sprayed on the lower side of leaves using a plastic

atomizer.
5. The inoculated leaves are arranged with their petioles buried in

m o i s t river sand in a plastic tray covered with a polyethylene bag
with sufficient moisture. They are kept in an incubator in the
d a r k i n i t i a l l y a n d l a t e r for a 12 h p h o t o p e r i o d at 25°C. The
disease symptoms will start developing after 7 days.
This procedure can be carried out on single plants grown in small

pots as well as on rooted detached leaves. The detached leaves are
arranged in a tray with river sand supplemented with Hogland solution.
The percentage infection could be estimated using the leaf diagrams
depicting known percentages o f damage caused b y rust (Fig. 3 ) .
F i g u r e 3. R u s t r e a c t i o n (%) o n g r o u n d n u t l e a v e s .
HRDF SDS no. 6 15

MP 8. Early and Late Leaf Spots of Groundnut
Symptoms
Early leaf spot. I t i s c a u s e d b y Cercospora arachidicola H o r i . It
develops small necrotic flecks, that usually have light to dark-brown
centers, and a yellow halo. The spots may range from 1 m m — 1 0 mm in
diameter. Sporulation is on the adaxial (upper) surface of leaflets.
Late leaf spot. I t i s c a u s e d b y Phaeoisariopsis personata (Berk & Curt)

V. Arx. It develops small necrotic flecks that enlarge and become light
to dark brown. The yellow halo is either absent or less conspicuous in
late leaf spot. Sporulation is common on the abaxial (lower) surface of
leaves. Comparisons of early and late leaf spots are in T a b l e 3.
T a b l e 3. T h e comparisons of early and lata leaf spot of g r o u n d n u t .
Character Early leaf spot Late leaf spot
Stage of o c c u r r e n c e Early infection Usually late infection
S h a p e of s p o t Circular to irregular Usually circular
Leaf s u r f a c e on w h i c h m o s t
s p o r e s a r e p r o d u c e d and Upper surface, Lower surface, in
their arrangement ' random concentric rings
C o l o r of spot on Light brown to black, Brown to black,

u p p e r leaf s u r f a c e tending t o w a r d s b r o w n tending t o w a r d s
w i t h some y e l l o w h a l o black
C o l o r of spot on
lower leaf s u r f a c e 1 Brown Black
' Important distinguishing features.
Control Measures
o A c r o p r o t a t i o n of c e r e a l - c e r e a l - g r o u n d n u t and b u r y i n g a l l
groundnut crop residues by deep plowing will reduce initial
inoculum. Adjust the date of sowing to avoid conditions
f a v o r a b l e for rapid d i s e a s e d e v e l o p m e n t .
o M u l t i p l e applications of a fungicide such as b e n o m y l , c a p t a f o l ,
chlorothalonil, copper hydroxide, mancozeb, or sulphur
fungicides may control early and late leaf spot (Smith 1 9 8 4 ) .
However, carbendazim (0.05%) controls both leaf spots very
effectively.
o Three sprays of 0.2% chlorothalonil at intervals of 10—15 days
starting 40 days after germination up to 90 days provides
e f f e c t i v e c o n t r o l to early and late leaf s p o t s , and r u s t .
o G r o w c u l t i v a r s t o l e r a n t to late leaf s p o t : ICGV 87160 or ICGV
86590.
16 HRDP SDS no. 6

The fungicidal control of diseases is more effective if a disease
forecasting system based on temperature and relative humidity during the
growing season, as has been developed in Georgia, is used. The
fungicides are applied w h e n the temperature and leaf w e t n e s s conditions
are favorable for disease development. Indiscriminate application of
fungicides to control early and late leaf spots results in nondesirable
effects. For e x a m p l e , use of excessive chlorothalonil for control of
f o l i a r d i s e a s e s i n c r e a s e s t h e s e v e r i t y o f Sclerotinia b l i g h t (Smith
1984).
MP 9. Screening for Early and Late Leaf Spots

The preliminary and advanced screening methodology discussed for rust is
also applicable for screening of leaf s p o t s . N o r m a l l y , leaf spots are
scored on t h e 1 to 9 scale (Table 4 and Fig. 3 ) .
Table 4. D e s c r i p t i o n o f leaf spots s c a l e ( 1 — 9 ) .
Leaf spot
score Description D i s e a s e s e v e r i t y (%)
1 No disease 0
2 L e s i o n s largely o n lower l e a v e s ; no defoliation. 1- 5
3 L e s i o n s largely on lower l e a v e s ; v e r y few lesions

on m i d d l e l e a v e s ; d e f o l i a t i o n o f some l e a f l e t s e v i d e n t o n
lower l e a v e s . 6- 10
4 L e s i o n s on lower and m i d d l e l e a v e s , but severe on lower

l e a v e s ; d e f o l i a t i o n of s o m e leaflets evident on lower l e a v e s . 1 1 - 20
5 L e s i o n s o n a l l lower and m i d d l e l e a v e s ; o v e r 5 0 %
d e f o l i a t i o n of lower l e a v e s . 2 1 - 30
6 L e s i o n s s e v e r e on lower and m i d d l e l e a v e s ; lesions

on t o p leaves b u t less s e v e r e ; e x t e n s i v e d e f o l i a t i o n of lower
l e a v e s ; d e f o l i a t i o n o f some leaflets e v i d e n t o n m i d d l e l e a v e s . 3 1 - 40
7 L e s i o n s on all leaves b u t less s e v e r e on top l e a v e s ;

d e f o l i a t i o n of all lower and some m i d d l e l e a v e s . 4 1 - 60
8 D e f o l i a t i o n of all lower and m i d d l e l e a v e s ; lesions s e v e r e on

t o p leaves and some d e f o l i a t i o n o f t o p leaves e v i d e n t . 6 1 - 80
9 D e f o l i a t i o n o f almost a l l leaves leaving b a r e s t e m s ; s o m e

leaflets m a y b e p r e s e n t , b u t w i t h s e v e r e leaf s p o t s . 81-100
Subrahmanyam et a l . (1980) discussed the procedure for screening

for the leaf spot diseases in the greenhouse. Plants are grown in
plastic pots (15-cm diameter) containing a mixture of soil and farmyard
m a n u r e (4:1 v / v ) . In each pot, two plants are grown.
Inoculum (conidia) of Phaeoisariopsis personata (late leaf

spot) is collected from incubated, infected, and detached leaves of the
susceptible cultivars. Conidia are suspended in sterile tap water
HRDP SDS no. 6 17

containing a few drops of wetting agent, 'Tween 8 0 ' . Before spraying the
inoculum concentration is adjusted to approximately 50 000 conidia mL-1
of solution.
Inoculation is carried out, first when seedlings are 30-day

old and the second time when plants are 50-day old.
Disease development is recorded at 28 and 42 days after

inoculation to evaluate the following parameters:
a. Defoliation. The number of leaflets on the main stem and the

number of abscised leaves are counted on each plant to calculate
defoliation percentage.
b. Leaf area damage. It is estimated for each leaf on the m a i n stem

in comparison to the diagram depicting the known percentage of the
area affected (Fig. 5 ) .
c. Infection frequency. The number of lesions on e a c h leaf of the

main stem is counted 2 8 days after i n o c u l a t i o n . T h e leaf area is
estimated using a leaf area m e t e r . Infection frequencies are
reported as number of lesions cm-2 of leaf a r e a .
d. Lesion diameter. The diameter of 10 randomly selected lesions are

measured on the leaves of the main stem.
e. Sporulation. Five leaflets are taken from each main stem 42 days
after inoculation and incubated on moist filter paper in petri
d i s h e s a t 25°C u n d e r c o n t i n u o u s i l l u m i n a t i o n i n a p e r c i v a l p l a n t
growth c h a m b e r for 5 d a y s . On the 6th d a y , the lesions are
examined under a stereoscopic-microscope (x20) to score the degree
of sporulation on a 5-point s c a l e .
f. Subject the percentage value of recorded data to arcsine/angular

transformation for analysis.
g. Rating description (5-point s c a l e ) .

1 No s p o r u l a t i o n
2 Very few spores
3 Moderate sporulation
4 More sporulation than score 3
5 Extensive sporulation
18 HRDP SDS n o . 6
1 2 3
4 5 6
7 8 9
Figure 4. Leaf spots damage (1 to 9 scale) on groundnut p l a n t s .

HRDP SDS no. 6 19

No 0.5% 1%
Damage Damage Damage
2% 5% 10%
Damage Damage Damage
35% 50%
20%
Damage Damage
Damage
Figure 5. L e a f a r e a d a m a g e (%) c a u s e d b y l e a f s p o t s o n g r o u n d n u t .
20 HRDP SDS n o . 6
MP 10. Use of Cob's Diagram for Percentage Leaf
Area Damage by Rust and Early and Late Leaf
Spots
A 1—9 s c a l e i s u s e d f o r s c o r i n g f o l i a r d i s e a s e s o f g r o u n d n u t a s i t s a v e s
time. To study the disease development and to assess the efficacy of
chemical applied, the Cob's diagram and percentage damage of leaves are
considered. T h e d i a g r a m for s c o r i n g r u s t and leaf spots in field
conditions can be used as follows:
1. T h e s c o r i n g f o r l e a f a r e a d a m a g e (%) i s d o n e 4 0 - 4 5 d a y s a f t e r p l a n t
emergence at intervals of 10 or 15 days on separate data sheets
(Annexures 1 and 2 ) .
2. F i v e t o ten p l a n t s a r e r a n d o m l y s e l e c t e d and l a b e l e d plot"1 for e a c h
genotype in each replication.
3. Observations of defoliation are taken on the main axis starting at
the first leaf node and moving upward.
4. The damage caused by each d i s e a s e (rust or leaf spot) is recorded
using the diagram in Fig. 2 or Fig. 4. The data are converted to
calculate percentage damage for each leaflet. In this way,
complete data are recorded on 5 plants giving percentage damage on
each leaflet at regular intervals of 15 days.
5. M e a n d i s e a s e d a m a g e (%) b a s e d o n t h e i n d i v i d u a l l e a f i n f e c t i o n i s
calculated.
Example. The following data were taken from an experiment. There
were e i g h t treatments with three replications w h e r e rust ( R ) , late leaf
spot (LS) and percentage defoliation (DEF) data w e r e recorded (Table 5 ) .
Table 5. The observations recorded for treatment 2 of replication 1,

plot 1.
Leaf area damage (%)
Leaf no. Rust Late leaf spot Defoliation (%)
1 — — 100
2 — — 100
3 — — 100
4 — — 100
5 50 5 -
6 50 4 -
7 50 3 —
8 50 - -
9 40 — -
10 20 — -
11 10 — —
12 5 — —
Mean 23% 1% 400/12 = 33%
HRDP SDS n o . 6 21
Explanation. The data showed that the first four leaves were
defoliated on most plants. The average rust infection on the remaining
p l a n t s i s 2 3 % ; w h i l e t h e m e a n for late leaf s p o t i n f e c t i o n i s o n l y 1%. I n
the same way, the mean of each treatment can be calculated and the
transformed value could be subjected to analysis and further
interpretation.
MP 11. Bud Necrosis

Bud necrosis disease (BND) is caused by two serologically distinct
v i r u s e s , bud necrosis virus (BNV) and tomato spotted w i l t virus ( T S W V ) .
BND was first recorded in Brazil in 1941, and significant crop losses by
this disease have been reported from Australia, India, and the USA (Reddy
1984a).
Symptoms
Initial symptoms are concentric rings or chlorotic spots on young

leaflets. Subsequently terminal bud necrosis occurs especially when day
t e m p e r a t u r e s e x c e e d 30°C. Plants infected at early stages are severely
stunted. Occasionally, necrosis may spread to the petioles and then to
the stem leading to death of the plant. Later infected plants may only
show bud necrosis on a few branches and axillary shoot proliferations may
be restricted to the terminal portion (Reddy et a l . 1 9 9 1 ) .
In early infection, pods are seldom produced. In late infections,

pod size is reduced, shriveled, and mottled with discolored testa. The
virus is not transmitted by seed; it is transmitted by thrips.
Control Measures
o Use resistant/tolerant cultivars: ICGS 11, ICGS 4 4 , ICGV 87141,

ICGV 87187, ICGV 87119, ICGV 87121, ICGV 8 7 1 6 0 , ICGV 8 7 1 5 7 , or ICGV
86590.
o C o n t r o l of v e c t o r (thrips).
o Adjust date of sowing to avoid the peak disease incidence.
o Sow groundnut at a high plant density and maintain a good plant

stand.
o Intercropping of groundnut with cereals, i.e., pearl millet will

restrict spread of the virus.
o Avoid groundnut cultivation adjacent to the crops that are

susceptible to BNV, such as green gram or black gram.
22 HRDP SDS n o . 6
MP 12. Groundnut Rosette
Three rosette diseases have been recognized. They are "groundnut
chlorotic rosette" ( G C R ) , "groundnut green rosette" ( G G R ) , and "groundnut
mosaic rosette" (GMR). GCR and GMR are predominant in eastern and
southern Africa, whereas GGR appears to be restricted to western Africa
(Reddy 1 9 8 4 b ) .
Symptoms
Groundnut chlorotic rosette (GCR) is characterized by general chlorosis,

with a few green islands on young leaflets. Early infected plants are
stunted, progressively producing small chlorotic, curled, and puckered
leaflets. Older leaflets are bright-yellow with dark-green patches.
Plants infected late, show typical leaf symptoms w i t h o u t the m a r k e d
stunting and bushy appearance (Reddy 1 9 8 4 b ) .
Groundnut green rosette (GGR) infected plants show mild and

narrow chlorotic streaks on young leaflets. The older leaflets are dark-
green and reduced in size with their margins rolled outward. Early
infected plants are stunted and bushy, whereas on late infected plants a
proliferation of axillary shoots may be observed (Reddy 1 9 8 4 b ) .
Control Measures
o Several long-duration cultivars with resistance to rosette are

currently available. T h e s e i n c l u d e R G 1 , R M P 1 2 , R M P 9 1 , K H 14-9
A, M 25-M 6 8 , and M 69- M 101. Short duration rosette
resistant cultivars are being developed.
o A p h i s craccivora is mainly responsible for the spread of rosette

disease. S p r a y o f e n d o s u l f a n 4 % d u s t w i t h 1 k g a . i . ha'1 o r
d e m e t o n - s - m e t h y l 72-96 mL a.i. ha-1 p r o v i d e e f f e c t i v e c o n t r o l for
aphids. It is essential to know the peak period of aphid migration
before application of insecticides.
o The eradication of volunteer groundnut plants is helpful to prevent

perpetuation of virus inoculum during the off-season.
o Early sowing and m a i n t e n a n c e of a good plant stand are helpful in

reducing the disease incidence.
HRDP SDS no. 6 23

MP 13. Peanut Clump
Peanut clump is caused by peanut clump virus (PCV). Early-infected
plants do not produce pods. Even in late-infected plants, yield losses
of up to 60% have been observed.
Symptoms
Affected plants are severely stunted, and the new quadrifoliates exhibit
mosaic mottling and chlorotic rings. Subsequently produced leaflets tur
dark green with faint mottling. Infected plants become bushy and produc
several flowers. Very few pods are produced on infected plants and the
size of pod is reduced (Nolt and Reddy 1 9 8 4 ) .
Control Measures
o Avoid sowing virus-infected seed.
o Use soil solarization for at least 70 days during summer months.
o Use a soil biocide such as carbofuran.
MP 14. Detection and Inoculation of Plant

Viruses
Virus detection requires a well-equipped laboratory and highly trained
technicians. Techniques for the detection of plant viruses have been
described in a manual prepared by the Virology Unit at ICRISAT Center.
Steps include serology, electron microscopy, and transmission techniques
(Reddy 1 9 8 7 ) .
Detection Techniques
Reaction on a set of hosts, and serological techniques are widely being

used for detection and assay of mechanically transmitted plant v i r u s e s .
Mechanical inoculation of plant v i r u s e s

Mechanical or sap inoculation is the application of plant extracts or
solutions containing viruses on the leaves of healthy plants. This is
done in such a way that the virus can enter the plant cells. Viruses
that are transmitted by aphids nonpersistently, those which multiply in
epidermal or mesophyll cells, and those that reach very high
concentration in plants are usually mechanically transmissible. The
sap-transmissible viruses usually produce mosaic, mottle or ring spot
symptoms. By contrast, viruses that are persistently transmitted by
insects are restricted to the xylem or phloem and are not mechanically
transmissible.
24 HRDP SDS no. 6

P r e p a r a t i o n of p h o s p h a t e b u f f e r (PB) 0.05 M pH 7.0
a. D i s s o l v e 7.08 g o f d i b a s i c p o t a s s i u m p h o s p h a t e ( K 2 H P O 4 . 3 H 2 O ) a n d
1.2 g o f m o n o b a s i c p o t a s s i u m p h o s p h a t e a n h y d r o u s ( K H 2 P O 4 . ) i n 5 0 0 m L
of distilled water. Make up the volume of the solution to 1000 mL
with distilled water.
b. A d d 1.56 m L o f 2 - m e r c a p t o e t h a n o l o r 0 . 7 5 m L o f t h i o g l y c e r o l o r 1.26
g of Na2SO3 to the above 1000 mL buffer. All of these are reducing
agents that retard the inactivation of the virus by oxidizing
enzymes, and thus preserve infectivity.
c. I t i s n e c e s s a r y t o s t o r e t h e b u f f e r a t 4°C. If this is not

p o s s i b l e , use freshly prepared and chilled buffer for each
inoculation.
P r e p a r a t i o n of plant e x t r a c t s
a. Collect 1 g foliage showing typical virus symptoms (leaves showing

initial symptoms are p r e f e r r e d ) .
b. Grind the tissue in 9 mL of cold phosphate buffer in a m o r t a r and

pestle. Keep the mortar and pestle on ice before u s e . Trituration
is done till all the leaves are finely ground.
c. The extract is filtered through two layers of cheese cloth. The

extract is ready for inoculation.
Procedure of inoculation
a. Expose plants to darkness before making the sap inoculation. It is

better to do the inoculation either early in the morning or late in
the afternoon (during summer m o n t h s ) .
b. Dust 600 mesh carborundum on leaves using a spray bottle.
c. Hold the leaf on the palm of your left hand. Now gently rub the
inoculum over the leaf s u r f a c e . This is done either using a cotton
swab or a piece of muslin cloth soaked with inoculum.
Precautions
Greater pressure and excessive carborundum dusted on the leaf surface may
lead to scorching of the tissue. Soon after i n o c u l a t i o n , the leaf
surface is washed with tap water and plants are covered with moistened
n e w s p a p e r s for o n e d a y . Wash hands after each inoculation either with
soap or trisodium phosphate solution. Carborundum dust should not be
inhaled as it can settle in the lungs. It takes at least 5 min after
dusting to settle the carborundum on the leaf surface.
HRDP SDS no. 6 25

Procedure for local lesion assay
Extracts from either tomato spotted wilt virus or peanut mottle virus
infected leaves are used. Several dilutions of the inoculum (1 : 10,
1:100, 1:1000) are applied using the half leaf t e c h n i q u e . An edge of the
leaf is p u n c t u r e d with a forceps to distinguish between the two primary
leaves inoculated. All treatments are randomized and usually eight
replications are used.
Procedures for g r a f t i n g
The majority of plant viruses are transmitted by grafting. There are

three types of graftings.
a. Approach grafting. In this system, the scion is not detached from

the mother plant. A splice is made on the scion and the stock and
both the surfaces are united.
b. Bud grafting. A small piece of stem tissue from the scion is

grafted on to the stock after making a splice to fit the scion.
c. Wedge grafting. A wedge is made in the stock and the scion is cut
to fit the wedge and inserted into the stock.
Enzyme-linked immunosorbent assay (ELISA)
This is by far one of the most widely used and sensitive serological
test. It permits analysis of several samples under identical test
conditions. The most simple form of ELISA is called the direct antigen
coating (DAC) procedure. This is suitable for detecting viruses that
reach high concentration in plant tissues, in seed, and in disease
surveys.
Procedure (Fig.6)
Step 1. Add viral antigens present in purified preparations and adhered
to the w e l l surface of the E L I S A plate by incubating the extracts for 1 h
a t 37°C. T h e p l a t e is w a s h e d three times in P B S - t w e e n a l l o w i n g 3-min
soaking for each wash. This will result in the attachment of virus to
the walls.
Step 2. Add a high dilution of crude antiserum (usually over 1 : 1000) and
i n c u b a t e a t 37°C f o r 1 h . Wash as in step 2, r-globulins are attached to
virus particles that are attached to the walls of the well.
Step 3. Add anti-rabbit Fc-specific gamma globulins enzyme (prepared in

goat's serum) conjugated with alkaline phosphatase. I n c u b a t e a t 37°C f o r
1 h. Wash with distilled water and PBS-tween as in step 2. Anti-rabbit

globulins are attached to the virus plate.
Step 4. A d d the substrate for alkaline phosphatase (normally P-nitro

phenyl phosphate is used) or the sodium penicillin in bromothymol blue.
26 HRDP SDS n o . 6
In case of a positive reaction the r-globulins attached to the antigens
attached to the well surface will react with gamma globulins. This will
facilitate the attachment of conjugated anti-rabbit Fc-alkaline
phosphatase anti-Fc reacts with the substrate and produces colored
hydrolysates. The intensity of color is measured at 405 nm that is
proportional to the concentration of viral antigens.
In case of a negative reaction, the specific gamma globulins are

not retained, thus the anti-rabbit Fc-specific globulins are washed away
during the washing. Since alkaline phosphatase is not retained, the
substrate will now become yellow.
A special ELISA reader is used to measure the absorbance at 405 nm

(for P - n i t r o p h e n y l p h o s p h a t e t h a t i s c o n v e r t e d i n t o n i t r o p h e n o l ) . In
case of sodium penicillin, the positive reaction shows light-green
initially which turns dark-yellow showing a strong positive reaction.
The c o m p o s i t i o n o f E L I S A b u f f e r s a r e :
a. Composition of carbonate buffer.
Na2CO3 1.59 g (0.02 M)

Na2HCO3 2.93 g (0.02 M)
Add chemicals to distilled water and m a k e up to 1 L. The pH of the

buffer should be 9.60.
b. Composition of phosphate buffer saline (PBS-tween).
NaCl = 8.0 g
K H 2 P O 4 = 0.2 g
N a 2 H P O 4 2H 2 O = 2.9 g or Na2HPO4 = 1.15 g
KCl = 0.2 g
T w e e n - 2 0 = 0.5 Ml
Dissolve the above chemicals in distilled water and make the volume
up to 1 L.
c. Composition of conjugate buffer.
P B S (T) = 500 m L
Polyvinyl pyrollidone = 10 g (2%)
Ovalbumin = 1 g (0.2%)
d. Substrate buffer (pH 9.8)
97.0 m L diethanolmine
800 mL distilled water
0.2 g NaNO3
Add HCl to obtain a pH 9.8 and make the volume up to 1 L.
HRDP SDS no. 6 27

Precautions
Never rinse new ELISA plates before coating with the antigen, use them as
they are supplied by the manufacturer. Thorough washing between each
step is required to remove the excess reagents, thus avoiding a
nonspecific reaction. Carbonate buffer is used for coating the a n t i g e n s .
T h e p h o s p h a t e s b u f f e r saline c o n t a i n i n g T w e e n - 2 0 (PBS-tween) is used for
washing ELISA plates between the steps. Antibodies and conjugated
gamma-globulins are diluted in a conjugate buffer and the substrate is
p r e p a r e d in a s u b s t r a t e b u f f e r .
Note. The details of PAC and DAS discussed in the 'Identification

and detection of legume viruses' are available in the Virology Unit
at ICRISAT.
Step 1 Step 2.
Take a new polyethylene Add antiser-
plate Add plant e x t r a c t , u m , incubate
incubate 1h at 37 C. 1h at 37 C.
(Hydrophobic action)
Wash 3 times in P B S - Rinse 3 t i m e s

Iween solution. Sook with P B S -
for 3 min. Tap plates tween. Dry
over a towel to dry. on towel.
r—globulins
Virus particles a t t a c h e d attached to
to walls. virus particles.
Step 3. Step 4.
Add e n z y m e - l a b e l e d Add sodium or
anti —rabbit r—glabulins potassium solt
Incubote at 37 C for of penicillin
1 h. with b r o m o t h y -
mol blue.
Wash with distilled Light—green

water ond P B S - weak positive
tween. reoction.
or
Anti rabbit r—glabulins Orange—yellow

with enzyme a t t a c h e d strong p o s i t i -
to virus. ve r e a c t i o n .
Figure 6. Direct antigen coating ELISA.
28 HRDP SDS no.6

Technical terns used in ELISA
PBS Phosphate buffer saline
Virus Transmissible agent possessing definite shape. They are

intracellular, and cannot be cultured on artificial
media. They can be seen only under an electron
microscope.
Antiserum Blood serum containing antibodies.
r-globulins Commonest type of antibodies in serum.

(IgG)
Anti-rabbit Antibodies raised animals in other than rabbits.

(IgG)
Antigen A substance that stimulates the production of an

antibody when introduced into animal tissues.
Serum A watery liquid that separates from coagulating blood.
(Source: Personal communication with Dr D.V.R. Reddy and Mr Sudarshan

Reddy, ICRISAT 1991).
MP 15. Bacterial Wilt of Groundnut

Symptoms
Young infected plants show sudden wilting of stem and foliage with leaves
on dead plants remaining green. In mature infected plants a gradual
decline causes the foliage to turn yellow. Infected plant roots become
discolored and are dead. Dying branches often curl to form a "shepherd's
crook". The disease can be identified by dark-brown spots in the xylem
and pith. The streaming masses of bacteria from cut ends of stems placed
in water can be seen (Gitaitis and Hammons 1 9 8 4 ) .
Control Measures
o A crop rotation with cereal may reduce the incidence of a bacterial

wilt.
o Use the seed produced in unaffected areas.
o The groundnut variety, Schwarz 21, has a high l e v e l of bacterial

wilt resistance.
o Follow plant quarantine regulations to prevent the disease being

introduced into new areas.
HRDP SDS no. 6 29

MP 16. Methods to Identify Plant Parasitic
Nematodes
The identification of nematodes requires collection, preservation, and
preparation of slides of nematodes for microscopic examination followed
by their identification.
A. E x t r a c t i o n from soil
Nematode infested soil samples are collected with a spade or sampling

tube from the vicinity of plant roots and stored in plastic bags in the
laboratory at a low temperature (15-25°C).
1. R e c o v e r y by d e c a n t a t i o n of a soil suspension
A d d a b o u t 5 0 0 c m 3 o f s o i l t o 3-4 L o f w a t e r i n a b u c k e t a n d s t i r
vigorously to break up the soil particles so that nematodes are released.
Leave the solution for 1 min to let the soil particles settle to the
bottom of the bucket, leaving the nematodes in suspension. Transfer the
suspension into another bucket through a nest of sieves with opening of
850 μm, 250 μm, and 38 μm (=20, 2 5 0 , and 400 mesh inch"1). Return the
third to the first bucket. Repeat the process, again by stirring,
leaving the soil to settle, and by decanting through sieves. Finally,
pass the water through sieve in the second bucket again. Collect the
nematodes from the sieve by washing them with a fine stream of water from
behind the sieve into a beaker.
2. U s e of d i f f e r e n t i a l c e n t r i f u g a t i o n w i t h a s o l u t i o n of high specific
gravity
P r e p a r e a s u s p e n s i o n of 100 cm3 s o i l in 600 mL of w a t e r and stir w e l l .

P o u r t h r o u g h a c o u r s e s i e v e (850 μm p o r e s i z e ) i n t o a c o n t a i n e r to r e m o v e
stones and plant particles. Now transfer the soil suspension to
centrifuge tubes and centrifuge the m i x t u r e at 1000 x gravity for 5 m i n .
Add a small amount of kaolin to the soil before centrifugation, if clay
content in soil is low. Pour off the supernatant, w h i p the upper part of
the centrifuge tubes to remove debris. Add a sucrose solution to the
tubes (490 g s u c r o s e d i s s o l v e d in 1 L of w a t e r ) . R e s u s p e n d s o i l in t h e
tubes and centrifuge as above. Let it settle in water.
B. P r e p a r a t i o n of slides
Prepare slides to examine the nematodes at high magnifications. This can

be done by making temporary slides or permanent slides.
1. Temporary slides
C o n c e n t r a t e the n e m a t o d e s in a small v o l u m e of w a t e r s u s p e n s i o n in a
small petri dish. Under a dissecting microscope, remove individual
specimens by using a long-pointed pick. Lift a nematode from the bottom
of the dish with the pick. When it is at the surface of water, pass the
pick under the specimen and lift it through the air-water interface. It
30 HRDP SDS n o . 6
will be held on the pick by surface tension. Then submerge the tip of
the pick in a drop of water on a slide or small vial to release the
nematodes from the pick. The drop should contain just enough fluid to
spread completely to the edges of a cover slip. Cover the drop with a
cover glass and seal the edges with melted paraffin, v a s e l i n e , or with
nail polish to prevent rapid evaporation. Rapidly moving specimens can
b e t e m p o r a r i l y i m m o b i l i z e d b y h e a t i n g t h e s l i d e b r i e f l y t o a b o u t 50°C o r
use of anaesthetics like 0.25% propylene phenoxital in water or in 1%
aqueous carboxymethyl cellulose.
2. Permanent slides
Fixation of nematodes. This requires fixation of specimen in aqueous
solution of 11% formalin plus 6% glycerin. Concentrate the specimens in
few milliliters of water in a v i a l . Bring the fixative to boil in a test
tube and rapidly add a small quantity of fixative to the v i a l , equal to
the amount of water in the vial. Cap the vial. The specimens are well
fixed after a few hours and are usually straight.
Specimens are placed in pure glycerin for p e r m a n e n t s l i d e s .

Lactophenol with a dilute concentration of cotton blue (0.0025%) is used
for semipermanent preparation. Add a drop of the tinted lactophenol to a
slide and w a r m it on a h o t p l a t e to 65-70°C. Transfer the specimens
directly from the fixative to the hot lactophenol and maintain the
t e m p e r a t u r e f o r 2-3 m i n . Then transfer them to nontreated tinted
lactophenol, cover and seal.
Infiltration with glycerin. Water contents of the fixed specimens are

gradually replaced to fix nematodes with pure glycerin. This could be
accomplished in few hours or over a long period. The procedure involves
the following:
a. Transfer the nematodes from the fixative to a small dish containing

0.5 m L s o l u t i o n o f 9 6 % e t h a n o l 2 0 p a r t s ; g l y c e r i n 1 p a r t ; d i s t i l l e d
water 79 parts.
b. Place the container with the nematodes in a desiccator containing
9 6 % e t h a n o l for 12 h at 35-40°C.
c. Remove the container with the nematodes, and fill with a solution
of 5 parts glycerin in 95 parts of 9 6 % e t h a n o l , p l a c e in a partly
c l o s e d p e t r i d i s h a n d m a i n t a i n a t 40°C f o r 3 h o r u n t i l t h e a l c o h o l
evaporates. Now the nematodes should be in pure glycerin. There
are several other procedures (Dropkin 1 9 8 0 ) .
Mounting. Pickup the nematodes individually and transfer them to a drop

of glycerin on a slide. Place a few glass fibers in the drop as spacers.
Add a cover glass and seal to prevent gradual loss of the fluid and
gradual absorption of moisture from the atmosphere.
C. E x t r a c t i o n from p l a n t s
The simplest way is to cut samples of infected plant parts (roots or
o t h e r p l a n t t i s s u e s ) i n t o 1-cm p i e c e s a n d p l a c e t h e m i n a n e l e c t r i c
blender together with water. After 20-30 sec of m a c e r a t i o n , the tissues
will be separated into small pieces, but the nematodes remain intact and
HRDP SDS no. 6 31

can be recovered from the suspension.
Another way is to use a mist chamber where the water is sprayed

intermittently from nozzles with small openings. The flow of water is
controlled by a solenoid and a timer to operate the spray about 1 0 % of
the time. Funnels are placed under the spray, each draining into a test
tube. The flow of water is very slow so that nematodes are washed into
the tube and remain at the bottom while aerated water slowly circulates
through the tube and flows out of the top. Both plant tissues and soil
may be extracted in a mist chamber. In cool conditions, the water must
be heated to avoid low temperatures during evaporation of the spray. The
extraction usually proceeds for up to 1 w e e k for c o m p l e t e recovery of
nematodes from roots or soil (Dropkin 1 9 8 0 ) .
Staining nematodes w i t h i n root tissues. This requires preparation of two

lots of lactophenol solution (phenol 20 g; lactic acid 20 g; glycerin 40
g; distilled water 20 m L ) . To one solution add 5 mL of a 1% aqueous
solution acid fuchsin (red) or cotton b l u e . Leave the other without dye.
Wash the root thoroughly in tap water. Bring tinted lactophenol to

boil in a well ventilated place, and boil roots in the staining solution
for 1 m i n . Wash out excess stain with tap water and blot off the excess
water. Place in clear lactophenol solution until the roots are
t r a n s l u c e n t i n 1-2 d a y s . Examine the roots in lactophenol or glycerin
under the dissecting microscope at 20X. The nematodes retain the dye
while the plant tissues are destained. Meristematic regions will also be
stained (Dropkin 1 9 8 0 ) .
MP 17. Root-knot Nematode

The root-knot nematodes (Meloidogyne spp.) are the m o s t important
nematode species causing damage ranging from 2 0 % to 9 0 % in infested
fields of groundnut (Rodriguez-Kabana 1 9 8 4 a ) .
Root galls contain white swollen adult females. The body tapers
anteriorly to a narrow neck and mobile head with stylet, massive median
bulb and large esophageal glands. An egg sac often protrudes posteriorly
from the female to the exterior of the gall. It contains several hundred
eggs. Often one or more elongate males are present in an egg sac. The
f e m a l e s a r e 0.5 m m t o 0.8 m m l o n g . At the center of its posterior
region, the female cuticle has a pattern of cuticular markings
surrounding the anus and vulva. The second stage of juveniles invade
roots at or close to the tip and migrate to the site of differentiating
vascular tissues. Consequently several giant cells form around the
nematodes head. The complete life cycle takes 3 weeks or m o r e , depending
on host and temperature. M a l e s a v e r a g e a b o u t 1.1 m m i n l e n g t h . The
posterior is characteristically twisted through 90 degrees or more.
Larvae are about 400 μm long and have a delicate stylet (Dropkin 1 9 8 0 ) .
32 HRDP SDS no. 6

Disease Symptoms
T h e s y m p t o m s o f d a m a g e c a u s e d b y Meloidogyne hapla a r e s i m i l a r t o t h o s e
c a u s e d b y M . arenaria. Root-knot nematodes enter and damage groundnut
roots, p e g s , and pods. Infected plants develop enlarged roots and pegs.
Galls develop into various sizes resulting from an internal swelling from
the root tissue. Infected pods develop knobs, protuberances, or small
warts. Infected plants with root-knot nematodes may show various degrees
of stunting and chlorosis. Root development is reduced, and vascular
systems of infected tissues are disrupted, resulting in the poor flow of
water a n d n u t r i e n t s f r o m t h e r o o t s (or p e g s ) t o t h e s h o o t . Infected
plants tend to wilt under drought conditions.
Control Measures
o A c r o p r o t a t i o n of c e r e a l - c e r e a l - g r o u n d n u t c a n s i g n i f i c a n t l y
decrease the level of root-knot nematode infestation in soils.
o Nematicides used in groundnut are fumigant and nonfumigant types

with contact or systemic properties. Application of a fumigant
nematicide like ethylene dibromide (EDB) is made 18 cm deep at a
s o i l t e m p e r a t u r e b e t w e e n 15—21°C @ 18 or 19 L h a - 1 . N o n f u m i g a n t
nematicides are aldicarb, carbofuran, and phenamiphos. These
n e m a t i c i d e s a r e e f f e c t i v e w h e n a p p l i e d a t s o w i n g @ 2-3 k g a . i . h a - 1 .
The best results are obtained when applications of nematicides are
m a d e i n a b a n d 1 7 - 2 5 c m w i d e a n d i n c o r p o r a t e d 2-4 c m i n t o t h e s o i l
(Rodriguez-Kabana 1984a).
o Soil solarization during the hot dry season, also helps to control
nematodes.
o Grow resistant cultivars: NC 343, NC 3033, NCAC 17090, or ICGS 2.
MP 18. Root-lesion Nematode

Root-lesion nematodes (Pratylenchus brachyurus) are small vermiform
nematodes. T h e a d u l t s a r e g e n e r a l l y l e s s t h a n 0.5 m m i n l e n g t h . Females
have a long slender stylet with rounded knobs, whereas males usually have
less developed or no stylets. Body annulations are fine. The vulva is
in the posterior portion of the body. There is a single ovary. The
procorpus and metacarpus are fused and the crescentic plates are large.
Disease Symptoms
Lesion nematodes are migratory endoparasites that attack groundnut roots,

pegs, and pods. They feed w i t h i n parenchymatous t i s s u e s . Both
mechanical and chemical damage result from the nematodes feeding within
the tissues. Root lesions develop and with large nematode populations
these lesions coalesce, causing extensive discoloration and damage that
results in reduced growth and pod production.
HRDP SDS no. 6 33

The pod lesions begin as tiny, tan to brown, pin-point areas on the
surface. As the nematodes feed and reproduce the affected area becomes
larger and darker. Old lesions are characterized by their blotchy
appearance and indistinct margins. This is caused by the darker
necrotic parenchyma, the outer cells of the pod and the necrotic areas
become diffused. Sometime nematodes are established in the roots without
visual symptoms above the ground, and cause yield reduction (Boswell
1984).
Control Measures
o A p p l y c a r b o f u r a n @ a . i . 4-8 k g h a - 1 i n t h e i n f e c t e d r o w s .
o Grow resistant genotypes such as PI 390606, PI 395233, or PI
365553.
o Solarize the soil during the hot dry season to control root lesion
nematodes. The soil temperature during solarization should rise
a b o v e 60°C t o k i l l t h e n e m a t o d e s a s w e l l a s s o i l b o r n e f u n g i .
MP 19. Ring Nematode

Ring nematodes ( M a c r o p o s t h o n i a xenoplax) are short and thick bodied
nematodes. They move slowly and cannot be collected from the soil in
Baermann funnels, but may be recovered after centrifugal flotation or by
migration from soil in shallow layers.
T h e females are fusiform and have less than 200 a n n u l u s e s . These

are broad, often overlapping, and have smooth, irregular posterior
margins of the annulations. They have a single outstretched ovary. The
vulva is located posteriorly, close to the anus. Males have longitudinal
incisures in lateral fields and candal alae. These nematodes are
ectoparasites, partly embedded in root tissues with a long stylet,
reaching w e l l into the root (Dropkin 1 9 8 0 ) .
Disease Symptoms
U s u a l l y only a large population of nematodes p r o d u c e symptoms of a

chlorotic appearance that have been called "peanut yellows." In
m i c r o p o t s inoculated with about 10 000 nematodes plants-1, the r o o t s ,
pods, and pegs of Argentine and Starr cultivars were found severely
discolored with brown necrotic lesions. Many root primordia and young
roots were killed, resulting in a few lateral roots. Pod yields from
nematode-infested plants were only 50% of the healthy plant yields
(Minton 1 9 8 4 ) .
Control Measures
o F o l l o w a c r o p rotation such as t o b a c c o - m a i z e - g r o u n d n u t to reduce
the population of nematodes,
o Fumigants and organophosphate nematicides are effective against
ring nematodes,
o Solarize the soil during the hot dry season.
34 HRDP SDS no. 6

MP 20. Testa Nematode
Testa nematodes ( A p h e l e n c h o i d e s arachidis) feed on fungi and parasitize
buds and leaves of the plants. They are long, slender nematodes with a
large metacarpus and blunt or pointed tails. The intestine joins
directly to the metacarpus and the esophageal glands overlap the
intestine in a long lobe. Males lack caudal alae and have a
characteristic thorn shaped spicule. Many species possess a sharp tip on
the tail called the mucron. The lateral fields have a few incisures
(Dropkin 1 9 8 0 ) .
Symptoms
Testa nematode is an endoparasite of groundnut. This causes

discoloration of seed tissues, reduces seed size, and causes seed
shriveling. This nematode occurs within the tissues of pods, testa,
roots, and hypocotyl. Infected groundnut seed coats are discolored when
m o r e than 2 000 nematodes testa-1 are present, often more than 25 000
nematodes are present. Heavily infected seeds, immediately removed from
fresh mature pods have a translucent testa. Infected testa of dry seed
are often wrinkled and dark brown. Infection also reduces the seed mass
and seedling emergence (Rodriguez-Kabana 1 9 8 4 c ) .
Control Measures
o Avoid sowing the infected seed.
o Soak the infected seed in cold water for 15 min, followed by a hot
w a t e r (60°C) t r e a t m e n t f o r 5 m i n .
o Dry the harvested pods in the hot sun or drier to a low moisture
content ( 8 % ) .
MP 21. Sting Nematode

Sting nematodes ( B e l o n o l a i m u s longicaudatus) are large slender nematodes
with an off-set head separated by grooves into four lobes. A stylet is
a b o u t 100 μm long. The procorpus has a long coiled lumen when the stylet
is retracted. There is a single incisure in the lateral field. Two
ovaries are present. The female tail is cylindrical and equal in length
to three times the body width at the anus, the terminus is round. The
caudal alae of the male are large. The esophageal glands overlap the
intestine (Dropkin 1 9 8 0 ) . Males and females of this nematodes are
morphologically similar. They range in length from 2 mm to 3 mm and have
a strongly striated cuticle (Rodriguez-Kabana 1984b).
HRDP SDS no. 6 35

Disease Symptoms
Affected plant roots become gnarled and stubby, with the tap root
frequently being the only root left over. Feeding results tiny lesions
along the roots, affected plants become chlorotic with stubby, sparse
root. Affected roots and pods have small, dark necrotic spots. They are
ectoparasitic and rarely found internally in roots and pods
(Rodriguez-Kabana 1984b).
Control Measures
Use of the nematicide, fensulfothion, provides effective control for

sting nematodes.
36 HRDP SDS n o . 6
References
Ainsworth, G.C., Sparrow, F.K., and Sussaan, A.S. 1973. The fungi. A
taxonomic review with keys. V o l . 4, A and B: N e w York, USAt Academic
Press.
Boswell, T.E. 1984. Root-lesion nematodes. Pages 41-42 in Compendium

of peanut diseases (Porter, D.M., Smith, D.H., and R o d r i g u e z - K a b a n a , R.,
eds.). St. Paul, MN, USA: American Phytopathological Society.
Dropkin, V.H. 1980. Introduction to plant nematology. New York, USA:

John Wiley.
Feakin, S.D. (ed.) 1973. Pest control in groundnuts. PANS Manual no.
2. London, UK: Centre for Overseas Pest Research.
Gibbs, A., and Harrison, B. 1976. Plant virology: The principles.

London, UK: Edward Arnold Publishers. 292 pp.
Gitaitis, R.D., and Hansons, R.O. 1984. Bacterial wilt. Pages 36-37 in
Compendium of peanut diseases (Porter, D.M., S m i t h , D.H., and R o d r i g u e z -
K a b a n a , R., e d s . ) . St. Paul, MN, USA: American Phytopathological
Society.
Mayee, C.D., and Datar, V.V. 1986. Phytopathometry. Technical Bulletin

no. 1. Parbhani, Maharashtra, India: Marathwada Agricultural
University. 146 p p .
Mehan, V.K., and McDonald, D. 1983. Screening for resistance to

Aspergillus flavus invasion and aflatoxin production in groundnuts.
Occasional Paper no. 2. Patancheru, A.P. 502 3 2 4 , India: International
Crops Research Institute for the Semi-Arid Tropics. 15 pp. (Limited
distribution.)
Minton, M.A. 1984. Ring nematodes. Pages 43—44 in Compendium of peanut

d i s e a s e s (Porter, D.M., Smith, D.H., and R o d r i g u e z - K a b a n a , R., e d s . ) .
St. Paul, MN, USA: American Phytopathological Society.
Holt, B.L., and Reddy, D.V.R. 1984. Peanut clump. Pages 50—51 in
C o m p e n d i u m of peanut diseases (Porter, D.M., Smith, D.H., and R o d r T g u e z -
Society.
Pettit, R.E. 1984. Yellow mold and aflatoxin. Pages 35—36 in

Compendium of peanut diseases (Porter, D.M., Smith, D.H., and Rodriguez-
Society.
Reddy, D.V.R. 1984a. Tomato spotted wilt virus. Pages 48—49 in

C o m p e n d i u m of peanut diseases (Porter, D.M., S m i t h , D.H., and R o d r i g u e z -
Society.
HRDP SDS no. 6 37

Reddy, D.V.R. 1984b. Groundnut rosette. Pages 49—50 in Compendium of
p e a n u t d i s e a s e s (Porter, D.M., Smith, D.H., and R o d r i g u e z - K a b a n a , eds.).
St. Paul, MN, USA: American Phytopathological Society.
Reddy, D.V.R. 1987. Techniques used for detection of plant viruses.

Lecture note to VII International Training Course in Legume Pathology,
12-31 Jan 1987, ICRISAT Center, India. Patancheru, A.P. 502 324, India:
International Crops Research Institute for the Semi-Arid Tropics.
(Limited distribution.)
Reddy, D.V.R., and McDonald, D. 1983. Pages i—viii in Proceedings of

the National Seminar on Management of Diseases of Oilseed Crops, 21—22
Jan 1983, M a d u r a i , India (Narayanasamy, P., e d . ) . Madurai, Tamil Nadu,
India: Tamil Nadu Agricultural University, Agricultural College and
Research Institute.
Reddy, D.V.R., N i g h t m a n , J.A., B e s h e a r , R.J., H i g h l a n d , B . , B l a c k , M.,

S r e e n i v a s u l u , P., D w i v e d i , S.L., D e m s k i , J.W., M c D o n a l d , D . , S m i t h J r . ,
J.W., and S m i t h , D . H . 1991. Bud necrosis: A disease of groundnut caused
by tomato spotted wilt virus. Information Bulletin no. 31, Patancheru,
A . P . 502 3 2 4 , India: International Crops Research Institute for the
Semi-Arid Tropics.
Rodrigues-Kabana, R. 1984a. Root-knot nematodes. Pages 38—41 in

Compendium of peanut diseases (Porter, D.M., Smith, D.H., and Rodriguez-
Society.
Rodrigues-Kabana, R. 1984b. Sting nematodes. Pages 42—43 in Compendium

of p e a n u t d i s e a s e s (Porter, D.M., S m i t h , D.H., and R o d r i g u e z - K a b a n a , R.,
Rodrigues-Kabana, R. 1984c. Other nematodes. P a g e 4 4 in. C o m p e n d i u m o f

peanut diseases (Porter, D.M., Smith, D.H., and R o d r i g u e z - K a b a n a , R.,
Smith, D.H. 1984. Early and late leaf spots. P a g e s 5—7 i n C o m p e n d i u m

of p e a n u t d i s e a s e s (Porter, D.M., Smith, D.H., and R o d r i g u e z - K a b a n a , R.,
S u b r a h m a n y a m , P., G i b b o n s , R.W., Nigam, S.N., and Rao, V.R. 1980.

Screening methods and further sources of resistance to peanut rust.
Peanut Science 7:253-256.
S u b r a h m a n y a m , P., M c D o n a l d , D . , and H a m m o n s , R . O . 1984. Rust. Pages

7—9 i n C o m p e n d i u m o f p e a n u t d i s e a s e s ( P o r t e r , D . M . , S m i t h , D . H . , and
R o d r i g u e z - K a b a n a , R., eds.). St. Paul, MN, USA: American
Phytopathological Society.
S u b r a h m a n y a m , P., a n d R a v i n d r a n a t h , V . 1988. Fungal and nematode

diseases. Pages 453—523 in Groundnut (Reddy, P.S., e d . ) . New Delhi,
India: Indian Council of Agricultural Research.
38 HRDP SDS no. 6

Annexure 1
Rust Scoring Data Sheet
E n t r y not Date:
Replication : Treatment :
Field not O b s e r v a t i o n not
Line no. Plot no. 1 Plot no. Plot

no. LAD (%) LAD (%)
LAD (%)
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
Total
Mean
1. LAD (%) = Leaf Area Damage (%)
HRDP SDS no. 6 39

Annexure 2
Leaf Spot Scoring Data Sheet
E n t r y no: Date :
Replication: Treatment :
Field no: Observation no:
1
Line no: Plot no. Plot no. Plot
no.
LAD (%) LAD (%) LAD (%)
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
Total
Mean
1. LAD (%) = Leaf Area Damage (%)
40 HRDP SDS no. 6

Evaluation
Select the m o s t a p p r o p r i a t e answer and check the correct a n s w e r at the
end of the b o o k l e t .
1. A n y d i s t u r b a n c e in the physiological processes of a plant is a

a) causal organism. b) disease,
c) pest. d) stress.
2. Nonchlorophyllous, nucleated, unicellular or multicellular

filamentous bodies that are reproduced by sexual or asexual spores
are
a) bacteria. b) virus.
c) fungi. d) microbes.
3. Biotrophs are active only in

a) nonliving host. b) living host.
c) vectors. d) soil.
4. The fungi that are mostly propagated by motile zoospore formed in

sporangia, without production of hyphae are
a) Ascomycetes. b) Basidiomycetes.
c) Deuteromycetes. d) Phycomycetes.
5. The unicellular organisms of very small cells that lack organized

nucleus are
a) fungi. b) viruses.
c) bacteria. d) mycoplasma.
6. Generally bacteria causing plant diseases are

a) gram positive. b) gram negative.
c) gram neutral. d) none of the above.
7. The submicroscopic organisms composed of protein and nucleic acid,

that multiply only in the host cell and do not possess an enzyme
system are
a) fungi. b) bacteria.
c) mycoplasma. d) viruses.
8. A vector is an agent which transmits

a) bacteria. b) fungi.
c) virus. d) mycoplasma.
9. The seed and seedling diseases caused by fungi in groundnut are

a) bud necrosis, mottling, and stripe.
b) root r o t , w i l t , and aflaroot.
c) rust and leaf spots.
d) root knot and stunting.
10. Groundnut rust is caused by

a) Cercosporidium personatum. b) Aspergillus niger.
c) Puccinia arachidis. d) Cercospora arachidicola.
HRDP SDS no. 6 41

11. Late leaf spot of groundnut is caused by
a) Cercospora arachidicola. b) Puccinia arachidia.
c) Cercosporidium personaturn, d) Aspergillus flavus.
12. Early leaf spot of groundnut is caused by

a) Aspergillus niger. b) Puccinia a r a c h i d i s .
c) Phaeoisariopsis personata. d) Cercospora arachidicola.
13. The vector of peanut clump virus is

a) Aspergillus niger. b) Aspergillus flavus.
c) Polymyxa graminis. d) Puccinia arachidis.
14. Bacterial wilt of groundnut is caused by

a) Pseudomonas solanacearum. b) Corynebacterium
insidiosum.
c) Xanthomonas tumefaciens. d) Agrobacterium tumefaciena.
15. Aflaroot or yellow mold in groundnut is caused by

a) Aspergillus niger. b) Aspergillus flavua.
c) Botrytis cineria. d) Fusarium solani.
16. Crown rot or collar rot of groundnut is caused by

a) Aspergillus niger. b) Aspergillus flavua.
c) Botrytis cineria. d) Fusarium aolani.
17. Stem rot of groundnut is caused by

a) Cylindrocladium crotalariae. b) Pythium myriotylum.
c) Sclerotinia minor. d) Sclerotium rolfsii.
18. In groundnut Rhizoctonia aolani and Pythium myriotylum cause

a) seed and seedling rot.
b) crown or collar rot.
c) damping o f f , root rot, and pod break down.
d) vascular w i l t .
19. Groundnut pod rots are caused by

a) F u s a r i u m solani and F. oxyaporum.
b) Macrophomina phaaeolina and Rhizoctonia aolani.
c) Sclerotium rolfaii and Aspergillua niger.
d) the combined attack of all the above.
20. In groundnut, the sudden wilting of lateral branches at an early

stage with the affected branches becoming chlorotic and turning
brown are symptoms of
a) blight. b) black rot.
c) stem r o t . d) vascular w i l t .
21. When germinating seeds are covered with masses of black conidia, or
there is rapid drying of infected plants with a shaded dark brown
color in the collar region, these are symptoms of
a) wilt. b) collar r o t .
c) blight. d) stem r o t .
42 HRDP SDS n o . 6
22. Preemergence death of seedlings with shrunken elongated dark-brown
areas on the hypocotyl are symptoms of
a) Rhizoctonia solani. b) Fusarium solani.
c) Aspergillus flavus. d) Verticillium alboatrum.
23. Wilting, curling, and chlorotic symptoms on leaflets and petioles

are due to an infection caused by
a) F u s a r i u m oxysporum. b) Aspergillus flavus.
c) Rhizopus arrhizus. d) Verticillium alboatrum.
24. Following a cereal-cereal-groundnut rotation, seed dressings, and

clean groundnut cultivation are helpful to control
a) foliar diseases. b) seed and seedling d i s e a s e s .
c) viruses. d) all the diseases.
25. When infected plants become stunted, with vein cleaning chlorosis
on the leaflets, and where seedlings lack secondary root symptoms,
the disease may
a) aflatoxin. b) toxin.
c) aflaroot. d) wilt.
26. The lines are identified as resistance to invasion and colonization

b y Aspergillus flavus w h e n s p o r u l a t i n g g r o w t h o f f u n g u s is up to
a) 11-20% of seeds. b) 1—16% of s e e d s ,
c) 15—25% of seeds. d) 30—50% of seeds.
27. The appearance of orange colored pustules (uredinia) in groundnut

plants and reddish brown uredospores on the abaxial surface of
leaves are symptoms of
a) early leaf s p o t . b) late leaf spot.
c) rust. d) virus.
28. The main symptoms of groundnut rusts are confined to the

a) roots. b) stems,
c) leaflet and aerial p a r t s . d) flowers.
29. Chlorothalonil is useful to control groundnut

a) e a r l y leaf spot. b) late leaf spot.
c) rust. d) all the above.
30. The nonreplicated test genotypes are grown with plants 60-cm apart
i n o n e - r o w p l o t s o f 4-m l e n g t h w i t h t h e i n f e c t o r r o w s f o r
a) advanced screening of rust.
b) p r e l i m i n a r y screening of rust and leaf s p o t s .
c) screening for virus.
d) screening for nematodes.
31. T e s t genotypes are replicated 2 or 3 times and each test p l o t is

separated by an infector row for
a) advanced screening of rust only.
b) advanced screening of rust and late leaf s p o t s .
c) screening for v i r u s .
d) screening for n e m a t o d e s .
HRDP SDS no. 6 43

32. For rust, infector rows are inoculated with urediniospore
suspensions of
a) volunteer plants. b) potted spreader plants,
c) infector row plants. d) natural infection.
33. For rust inoculation in the laboratory, spores are collected in a

glass tube with the help of
a) a sucking pump. b) the hand.
c) a cyclone spore collector. d) none of the above.
34. For inoculation purposes the rust inoculum is adjusted to

s p o r e s mL-1 of s o l u t i o n .
a) 20 000 b) 30 000
c) 40 000 d) 50 000
35. The spores in the inoculum are counted using a

a) cyclone spore collector, b) microscope,
c) hemocytometer. d) hygrometer.
36. After rust inoculation the disease symptoms start developing in

a) 3 days. b) 5 days,
c) 7 days. d) 9 days.
37. Leaf diagrams with a known percentage of disease damage are useful
for estimation of
a) leaf a r e a d a m a g e . b) percentage of infection.
c) disease severity. d) disease intensity.
38. Hogland solution is useful for

a) inducing disease infection. b) reducing disease infection.
c) enhancing plant growth. d) inducing root development.
39. Small necrotic flecks with a light to dark-brown center and a

yellow halo are the symptoms of infection caused by
a) e a r l y leaf s p o t , b) late leaf s p o t ,
c) rust. d) virus.
40. Small necrotic flecks that enlarge and become light to dark brown
without a y e l l o w halo and sporulations on the abaxial surface of
leaves are symptoms of infection caused by
a) rust. b) early leaf s p o t ,
c) late leaf spot. d) virus.
41. E x c e s s i v e u s e of c h l o r o t h a l o n i l for the c o n t r o l of foliar diseases

increases the severity of
a) Rhizoctonia blight. b) wilt.
c) viruses. d) Sclerotinia blight.
42. A s c o r e o f 1 o n a 1—9 s c a l e f o r l e a f spots or r u s t in groundnut

leaf disease severity is equal to damage,
a) 0% b) 1-2%
c) 2-3% d) 4-5%
44 HRDP SDS n o . 6
43. W h i l e scoring on 1 to 9 scale for leaf spot or rust disease
s e v e r i t y (%) i n g r o u n d n u t a s c o r e o f 3 i n d i c a t e s damage.
a) 1-5% b) 6-10%
c) 11-20% d) 21-30%
44. Estimation of each leaf damage in comparison to the diagram

depicting the known percentage of the area of late leaf spot damage
is used for calculating
a) defoliation. b) leaf area d a m a g e .
c) infection frequency. d) sporulation.
45. The number of late leaf spot lesions cm-2 of leaf area is
a) leaf area d a m a g e . b) defoliation.
c) infection frequency. d) lesion diameter.
46. Concentric rings or chlorotic spots on young leaflets and

subsequently terminal bud necrosis is caused by
a) wilt. b) peanut stripe virus,
c) tomato spotted wilt virus. d) iron chlorosis.
47. The vectors of tomato spotted wilt virus are

a) aphids. b) jassids.
c) thrips. d) spodopteras.
48. General chlorosis with a few green islands on young leaflets,

stunting of p l a n t s , and small chlorotic, curled, and puckered
leaflets are caused by
a) groundnut green rosette. b) groundnut mosaic rosette,
c) groundnut chlorotic rosette. d) peanut stripe virus.
49. The symptoms of narrow chlorotic streaks on dark green young

leaflets, a reduced leaf size with upward rolled margins of older
leaves are caused by
a) groundnut chlorotic rosette. b) groundnut mosaic rosette,
c) peanut stripe virus. d) groundnut green virus.
50. Groundnut rosette resistant cultivars are

a) ICGS 11 and ICGS 4 4 . b) RG 1 and 69-M 101.
c) TMV 2 and JL 2 4 . d) ICG(FDRS) 4 and ICG(FDRS) 10.
51. The insect vectors of groundnut rosette virus are

a) jassids. b) thrips.
c) aphids. d) spodopteras.
52. Symptoms of severe stunting of the plant with mosaic mottling,

chlorotic rings, and a bushy appearance of infected plants are
caused by
a) groundnut rosette virus. b) peanut stripe virus,
c) tomato spotted wilt virus. d) peanut clump virus.
53. Peanut clump virus is transmitted by

a) Aphis craccivora b) Scirtothrips dorsalis.
c) Polymyxa graminis. d) n o n e of t h e s e .
HRDP SDS no. 6 45

54. The best way to detect plant virus is
a) mechanical inoculation. b) local lesion assay.
c) grafting. d) ELISA assay.
55. The application of virus extracts on to the leaves of healthy plant

to facilitate virus infection is
a) grafting. b) ELISA.
c) mechanical inoculation. d) local lesions assay.
56. The viruses transmitted by insects are restricted to

a) leaves. b) stem.
c) roots. d) xylem and phloem.
57. When a substance introduced into animal tissue stimulates the

production of an antibody it produces
a) a virus. b) an antiserum.
c) an antigen. d) a fungi.
58. A watery liquid that separates from coagulating blood is

a) an antigen. b) an antibody,
c) a virus. d) a serum.
59. The sudden wilting of the groundnut stem and foliage with gradual
yellowing and curling of drying branches to form a shepherd's
crook, with dark spots in the xylem and pith is caused by
a) fusarium wilt. b) tomato spotted wilt virus,
c) root rot. d) bacterial wilt.
60. A bacterial wilt resistant variety of groundnut is

a) RG 1. b) ICGS 11.
c) ICG(FDRS) 4. d) Schwarz 21.
61. An elongated tubular unsegmented organism that looks like thread is

a) fungi. b) bacteria,
c) nematode. d) virus.
62. The root-knot nematodes can cause damage ranging from

a) 1-5%. b) 5-10%.
c) 10-15% d) 20-90%
63. The nematode affected roots, pegs, and pods develop into galls
caused by
a) root-lesion nematode. b) sting nematode.
c) root-knot nematode. d) ring nematode.
64. The m o s t appropriate time for application of nematicide in

nematode-infested field is
a) seed treatment. b) after sowing.
c) at the time of sowing. d) after infection is noticed.
65. The best result of nematicide application in affected areas is

obtained by
a) broadcasting. b) mixing with seed,
c) band application 2 cm to 4 cm deep. d) foliar spraying.
46 HRDP SDS no. 6

66. One of the management practice useful for the control of nematodes
is
a) deep plowing. b) shallow plowing.
c) deep irrigation. d) soil solarization.
67. The nematode which is a migratory endoparasite and can attack on

groundnut roots, pegs, p o d s , and feed within parenchymatous tissue
is
a) root-knot nematode. b) root-lesion nematode.
c) ring nematode. d) testa nematode.
68. The nematode that can develop in side root without visual symptoms
above the ground and cause yield reduction is
a) root-knot. b) root-lesion,
c) ring. d) testa.
69. Root lesion nematode resistant genotypes are

a) ICGS 11 and ICGV 76. b) TMV 2 and JL 2 4 .
c) PI 390606 and PI 395233. d) CS 9 and ICG(FDRS) 10.
70. The soil temperature during solarization to kill nematodes should

rise above
a) 30°C. b) 40°C.
c) 50°C. d) 60°C.
71. The short and thick bodied nematodes that move slowly and can not
be collected from the soil in Baermann funnels, but can be
recovered after centrifugal flotation is
a) root-knot nematode. b) root-lesion nematode.
72. The chlorotic symptoms called peanut yellows are caused by the
infection due to
a) root-lesion nematode. b) root-knot nematode.
c) testa nematode. d) ring nematode.
73. The nematode that feeds on soil fungi and parasitize on buds and
leaves of the plant is
a) root-lesion nematode. b) testa nematode.
74. Discoloration of groundnut seed tissues, reduction in seed size,

and shriveling of seeds are caused by
a) root-lesion nematode. b) root-knot nematode.
75. Cold and hot water seed treatment can kill

a) root lesion nematode. b) testa nematode.
76. Gnarled and stubby appearance of affected plants with barely left
over tap roots showing necrotic spots are the symptoms caused by
a) sting nematode. b) testa nematode,
c) ring nematode. d) root- knot nematode.
HRDP SDS no. 6 47

Correct responses to the questions.
1. b ) ; 2. c); 3. b ); 4. d ) ; 5. c ) ; 6. b ); 7. d ); 8. c ) ; 9. b);
10. c); 11. c); 12. d); 13. c); 14. a); 15. b); 16. a); 17. d);
18. c); 19. d); 20. c); 21. b); 22. a); 23. d); 24. d); 25. c);
26. b); 27. c); 28. c); 29. d); 30. b); 31. b); 32. b); 33. c);
34. d>? 35. c); 36. c); 37. b); 38. d); 39. a); 40. c); 41. d);
42. a); 43. b); 44. b); 45. c); 46. c); 47. c); 48. c); 49. d);
50. b); 51. c); 52. d); 53. c); 54. d); 55. c); 56. d); 57. c);
58. d); 59. d); 60. d); 61. c); 62. d); 63. c); 64. c); 65. c);
66. d); 67. b); 68. b); 69. c); 70. d); 71. c); 72. d); 73. b);
74. d); 75. b); 76. a).
48 HRDP SDS no. 6

Major Diseases of Groundnut PDF

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M a j o r Diseases of Groundnut

Faujdar Singh and D.L. Oswalt

Skill Development Series no. 6

Human Resource Development Program

Faujdar Singh and D.L. Oswalt

Skill Development Series no. 6

Human Resource Development Program

Publications in this Skill Development Series (SDS) are issued

C o m m e n t s , s u g g e s t i o n s , and e n c o u r a g e m e n t f r o m D r s D.V.R. R e d d y , D.H

MP 1. Seed Rots and Seedling Diseases of Groundnut 9

MP 9. S c r e e n i n g for E a r l y and L a t e Leaf Spots 17

MP 13. Peanut Clump 24

MP 17. Root-Knot Nematode 32

MP 21. Sting Nematode 35

Fungi are nonchlorophyllous, nucleated, unicellular, or multicellular

Myxomycota. This group includes unicellular fungi that produce

Kumycota. This group consists of filamentous fungi. These are

Mastigomycotina. They produce asexual spores in sporangia. They

Zygomycotina. T h e s e i n c l u d e Rhizopus spp. and Mucor spp. The

Asconycotina. These fungi destroy foliage and parts of plants that

Basidioaycotina. These include rusts and smuts that are host

Deuteromycotina, or Fungi imperfecti. In these fungi the sexual

HRDP SDS no. 6 5

o They are composed of protein and nucleic acid.

o They contain one type of nucleic acid, either RNA, or DNA.

o They multiply only in the host cell (obligate pathogens).

o Viruses do not possess enzyme systems, required to perform

o They are transmitted by graft inoculations, by sap, by

An agent t h a t transmits a virus is called a vector. In many

Nematodes (Greek for t h r e a d ) are e l o n g a t e , tubular organisms that m o v e

6 HRDP SDS no. 6

To protect a crop from diseases, it is important to know the causal

Figure 1. A plant parasitic nematode. (Source: Dropkin 1980)

HRDP SDS no. 6 7

Foliar diseases. The major foliar diseases of groundnut caused by

Bacterial wilt of groundnut caused by Pseudomonas solanacearum E.F. Sm.

Several nematodes are parasitic to groundnut. These are root-knot

8 HRDP SDS no. 6

T a b l e 1. P u n g i c a u s i n g seed rots and seedling d i s e a s e s of g r o u n d n u t .

Fungi Disease Symptoms

Aspergillus flavus Aflaroot or Affected seeds are shriveled and dried,

Fusarium solani and Wilt L o w e r end o f t a p r o o t b e c o m e s b r o w n t o

Rhizopus arrhizus and Seed and Sudden w i l t i n g of lateral b r a n c h e s that

Rhizoctonia solani Root rot, pod P r e e m e r g e n c e death of s e e d l i n g s ;

Pythium ultimum and Damping off Soft rot of the h y p o c o t y l r e g i o n c a u s i n g

Verticillum alboatrum Vascular wilt W i l t i n g o f leaflets a n d p e t i o l e s ,

o Follow a crop rotation, i.e., cereal-cereal-groundnut.

o Sow good quality and disease-free seed.

o Avoid damage to the seed testa and deep placement of seed at

o Treat the seed with thiram e 3 g kg-1 seeds or with carbendazim % 2

HRDP SDS no. 6 9

The occurrence of pod rots may be severe due to attacks of

o Use cereal-cereal-groundnut crop rotation and seed treatment with

o Avoid drought at pod formation and maturity.

o Avoid heavy irrigation before harvesting. It will lead to pod rot

MP 3. Groundnut Yellow Mold

Aflatoxins a r e c a r c i n o g e n i c a n d p r o d u c e d b y t h e Aspergillus flavus

Yellow mold first appears on groundnut cotyledons after the emergence of

10 HRDP SDS no. 6

o Dry the groundnut pods to 6-8% moisture content immediately after

harvesting and discard the infected pods and seeds.

o Avoid damage to the testa while decorticating.

o Prevent drought stress, and also prevent water logging (40-80% of