Fungal, bacterial and virus
contaminants of spices and spice
products
Collected by:
Sahab AL zamil 432200456
Noura ALsagheir 432201382
Amani ALmutairi 431201070
ALanoud ALshelali 432200859
Index:
Title.
Page No.
Introduction
History
Fungal introduction
Bacterial introduction
Viral introduction
Benefits of spices
Fungal materials and methods
Bacterial materials and methods
Viral materials and methods
Results
Fungal discussion
Bacterial discussion
Viral discussion
References
3
3
3
3
3
3-4
4
4-5
5
6-8
8-10
10-11
11-12
12
2
Introduction:
spread spice trade revolved around cinnamon and pepper throughout the Middle East as early
as 2000 B.C. The Egyptians fueled the need for exotic spices for embalming use and helped to
stimulate world trade. The Chinese and Indians in 1000 B.C. had a system of medicine based on
herbs also.Spices are valued for their distinctive flavors, colors and aromas and are among the
most versatile and widely used ingredient in food preparation and processing throughout the
world. As with many other agricultural products, spices may be exposed to a wide range of
microbial contamination during pre- and post-harvest. such contamination may occur during
processing storage, distribution, sale and/or use (McKee, 1995). Early cultures also recognized the
value of using spices in preserving foods and for their medicinal value. Spices have been used in
many industries, with the food industry and catering being predominant users. Having been dried
material from plant origin, spices are commonly heavily contaminated with xerophilic storage
molds and bacteria (Dimić et al., 2000; Romagnoli et al., 2007). Although spices are present in
foods in small amounts, they are recognized as important carriers of microbial contamination
mainly because of the conditions in which they were grown, harvested and processed. most such
microbial populations are probably regarded as commensal residents on the plant that survived
drying and storage. Soil and air is the main inoculum source for causing contamination in crude
spices in field. Other practices like harvesting, handling and packing, cause additional
contamination. Moreover, spices are collected in tropical areas by simple methods and are
commonly exposed to many contaminants before, being dry enough to prevent microbial growth.
The most frequent fungal contaminants of
spices are species from the genera
Aspergillus and Penicillium (Silliker et al.,
1992; Dimić and Škrinjar, 1995). ). Some
species that belong to these genera are
known as potential producers of different
toxic substances such as aflatoxins,
ochratoxins and sterigmatocystine, i.e.
mycotoxins that exhibit toxic, mutagenic,
teratogenic and carcinogenic effects in
humans and animals (Frisvad et al., 2005;
Zinedine et al., 2006).
The bacteria species isolated from the
spices varied. The spices and spice products
collectively
harboured
Aeromonas
salmonicida,
Enterobacter
cloacae,
Enterobacter
amnigenus,
Enterobacter
agglomerans,
Enterobacter
sakazakii,
Flavobacterium
sp,
Chromobacterium
violaceum, Pseudomonas putida, Pseudomonas
aeroginosa, Acinetobacter sp, Pseudomonas
cepacia, Serratia plymuthica. .
Piper yellow mottle virus (PYMoV) (genus:
Badnavirus) is known to infect black pepper,
the virus induces chlorotic mottling, vein
clearing, leaf distortion, reduced plant vigor
and poor fruit set in affected black pepper
plants.
Rosemary is used for culinary purposes
and in cosmetics. Studies carried out in the
last several years showed that oil from the
leaves of Rosemary can help prevent the
development of cancerous tumours in
laboratory animals (Anon, 2006). Maggi onion
contains iodised salt, flavour enhancers,
starch, vegetable fat, sugar, aniseed, cloves,
onion and other spices while Maggi shrimp
contains iodised salt, flavour enhancers,
starch, vegetable fat, sugar, shrimp powder
and spices (not specified). As a trade secret,
the consumer does not know the exact
compositions of the ingredients
The black pepper (Piper nigrum L) vine
and its extracts have been used as a folk
medicine in a variety of cultures and are the
source of the most commonly used spice
worldwide. Vitamins content in black pepper:
choline, Folic acid, Niacin, Pyridoxine,
Riboflavin, Thiamin, Vitamin A, and Vitamin C
Vitamins A, Vitamin E, Vitamin K. Minerals
3
content of black pepper: Calcium, Copper,
Iron, Magnesium, Manganese, Phosphorus and
Zinc.
Cinnamon has components that are thought to
offer some health benefits. It contains
antioxidants called proanthocyanidins and other
active
ingredients,
contains
proteins,
carbohydrates, vitamins (A, C, K, B3), Minerals
like Calcium, Iron, Magnesium, Manganese,
Phosphorous, Sodium, Zinc, Choline, Medicinal
uses: Lowers Cholesterol, Reduces blood sugar
levels, Fights Cancer, Cures Respiratory
Problems , digestive Tonic and Brain Tonic.
health benefits of Aniseed: diuretic, treatment
of digestive problems, relieves pain and oil of
anise has been used for the treatment of : Lice,
scabies and psoriasis.
Materials and methods:
Fungi:
2.1. Sampling:
About 138 samples of 15 different spices were collected randomly from famous supermarkets.
Samples (100 g/sample) were collected in sterilized polyethylene bags and stored at −4 °C until
use.
2.2. Mycological studies:
Dilution method according to Koch (Harrigan, 1998) was used to determine total fungal counts in
spice samples, in triplicates. Ten grams of each sample (fine powder) were added to 90 ml portion
of sterile saline solution (0.85%) in 500 ml Erlenmeyer flask and homogenized thoroughly on an
electric shaker at constant speed for 30 min. The spice–water suspension was allowed to stand for
10 min with intermittent shaking before being plated. Ten fold serial dilutions were then prepared.
One millilitre portion of suitable dilutions was used to inoculate Petri dishes containing 15 ml agar
medium fortified by 0.5 mg chloramphenicol/ml medium. Three nutritive media were chosen:
Czapek dox agar (dextrose, 10 g/L; NaNO3, 2.0 g/L; KCl, 0.5 g/L; MgSO4·7H2O, 0.5 g/L; FeSO4·7H2O,
0.01 g/L; K2SO4, 0.35 g/L; agar, 15.0 g/L; pH = 6.8 ± 0.2); potato dextrose agar (potatoes infusion,
200 g/L; dextrose, 20 g/L; agar, 15 g/L; pH = 5.6 ± 0.2) and Cooke rose (soytone, 5 g/L; dextrose,
10 g/L; KH2PO4, 1 g/L; MgSO4·7H2O, 0.5 g/L; agar, 20 g/L; rose bengal, 0.035 g/L; pH = 6.0 ± 0.2).
Plates were incubated at 28 ± 1 °C for 5–10 days and examined for the growth of moulds. Fung
were isolated and identified according to (Raper and Fennel (1977), Domsch et al. (1981), Pitt
(1985).
Bacteria:
3.1. Source of spice samples:
Collection of the samples: (aniseed, rosemary, maggi onion cube, maggi shrimp cube, royco shrimp
cube, royco beef cube, cinnamon, black pepper and masala).
3.2.Quantitative estimation of total aerobic bacteria
The initial bacteria population in the spices was determined by transferring 10 g samples into 250 ml
Erlenmeyer flasks containing 100 ml of 0.1% peptone water as diluents. Each flask was shaken at
140 RPM for 20 min on an orbital shaker (Gallenkamp, England). Serial dilutions up to 1: 104 were
made and 1 ml aliquots were plated on 20 ml PCA (plate count agar, BIOCHEMIKA 70152).
They were incubated at 35°C for 48 h. Colonies of bacteria that appeared were counted and
calculated as log10 CFU/g sample.
3.3..Isolation and identification of bacteria:
4
Bacteria colonies that developed on PCA were sub-cultured on nutrient agar until a pure culture
was obtained. These pure cultures were used for identification using an analytical profile index
(API) 20E kit (bioMérieux). Reactions were read according to a standard reading table and
identification was done using the analytical profile index book.
Virus:
Seeds collected from PYMoV infected plants (identified by PCR) of four varieties (IISR-Sreekara,
IISR-Subhakara, IISR-Shakthi and Panniyur-1) were sown in separate seed pans in the insect proof
glass house. Seeds collected from healthy plants (PCR negative plants) were also sown to serve as
controls. Seedlings were planted in separate polythene bags after three months of germination and
kept under insect proof condition for symptom development. For symptom observation, all the
available seedlings were used. To detect the presence of PYMoV in the berries and seedlings, PCR
test (based on total DNA as template) was used. Total DNA was isolated from tissues using the
protocol of Hareesh and Bhat (2008). Isolated total DNA was subjected to PCR using primers
specific for open reading frame (ORF) I / ORF III region of PYMoV . The PCR reaction (50 μl)
contained 50 ng each of the primers, 1.5 U Taq DNA polymerase , 1x PCR buffer, 250 μM MgCl2 and
10 μM each of the dNTPs and 31.5 μl of sterile water. PCR mix (45 μl) containing the above
components was added to the tubes containing the template DNA (5 μl) resulting in a final reaction
volume of 50 μl. The Seed transmission of PYMoV temperature profile for the reaction involved the
initial denaturation at 94 oC for 5 min followed by 35 cyles of denaturation at 94 oC for 30 s,
annealing at 50/56/58 oC (depending on the primers used for 1 min, synthesis at 72 oC for 1 min
followed by final extension at 72 oC for 10 min. During DNA isolation and PCR, two controls were
always used along with the test samples. These control samples included, a healthy virus free
sample (negative control) and a known PYMoV infected sample (positive control). The PCR results
were analyzed on 1 % agarose gel. The samples positive for PYMoV was identified based on the
presence of expected size fragment obtained in the reaction Initially to confirm the identity of
amplicons, the PCR products obtained using all the three sets of primers were purified and directly
sequenced at the automated sequencing facility . In order to find out the presence of PYMoV
particles (virions) in berries and seedlings, they were subjected to immunocapture (IC) PCR.
PYMoV specific polyclonal antibodies developed at Indian Institute of Spices Research was used at
1:1000 dilution to capture the virus particles. A 200 μl of diluted antibody was dispensed into 0.2
ml PCR tubes and incubated in a moist chamber at 37 0C for 2.5 h. Tissues were ground using
sterile mortar and pestle in antigen extraction buffer (20 mM Tris, 138 mM NaCl, 1 mM PVP, 3 mM
NaN3 and 0.05 % Tween 20) at a ratio of 1:5 (w/v)) at room temperature followed by
centrifugation at 8000 rpm for 30 s. The supernatant (200 μl) was added to the PCR tube after
discarding and washing of the antibody solution. The unbound antigens were washed off with PBST after 2.5 h of incubation. After washing, the tubes were added with PCR components and
subjected to thermocycling using three sets of primers as indicated above. The PCR products were
analyzed on 1 % agarose gel. Identity of the amplicons was confirmed by direct sequencing of the
PCR product at the automated sequencing facility
5
Results:
Total count of detected fungi in spices on different growth media figure 1
Heavy contaminated spice with fungi (ginger, 5325–6800 cfu g−1) on different media, coke rose (left), PDA
(middle) and Czapek (right).figure 2
Low contaminated spice with fungi (cloves, 163–300 cfu g−1) on different media, coke rose (left), PDA
(middle) and Czapek (right).figure 3
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No fungal contamination appears from sumac indicating its antifungal properties on the three media. Figure 4
Table 1. List of bacterial species isolated from raw spices and spice products plated on PCA at 35 to 37°C FOR 48
hours
Table 2. Prevalence of C. sakazakii in spices purchased from four
retail outlets
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Table 3. Effect of temperature treatments on black pepper inoculated with C. sakazaki.
Table 4. Symptoms and PCR test for the detection of Piper yellow mottle virus in seedlings of different varieties of
black pepper .
figure 4. Symptoms observed on black pepper seedlings raised from berries collected from Piper yellow
mottle virus infected plant (a) mosaic (b) chlorotic specks
Discussion:
In our study, most of the identified fungi have been reported to have the ability to produce
mycotoxins
(Bugno
et
al.,
2006).
In
this
context, Aziz
(1987) found
that, A. flavus, Aspergillus parasiticus andAspergillus oryzae were
aflatoxin
producers,
whereas, A. ochraceus, Penicillium viridicatum andPenicillium variable were
ochratoxin
A
producers.
In
addition, P. viridicatum, Penicillium chrysogenumand Penicillium commune were
penicillic acid producers. According to Pohland and Wood (1987), 70–80% of the penicillia are
potential mycotoxins producer (citrinin, patulin, cyclopiazonic acid and peniterm). These results
showed that a potential risk for mycotoxins contamination may be caused as a result of using these
spices, especially in great quantities. Rani and Singh (1990) found that 89% of samples of fennel,
coriander and cumin were contaminated with aflatoxin B1 at the levels 3000, 1640 and
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1580 mg/kg, respectively. In addition, Roy et al. (1988) and Roy and Chourasia (1990) determined
that the seeds of P.nigrum and Mucuna prurians, and the barks of A. catechu, C. sativum,
and Elettaria cardamomun were contaminated with aflatoxin B1 at levels below 20 mg/kg. Aziz and
Youssef (1991) isolated A. flavus andA. parasiticus with a high tendency for aflatoxin production
from some common herbal drugs and spices.Aziz et al. (1998) studied contamination of some
common medicinal plant samples and spices and their mycotoxins. Ten fungal genera
viz. A. flavus, A. parasiticus, A. niger, F. oxysporum and P. viridicatumoccurred most often on the
medicinal plant samples. Direct determination of mycotoxins in medicinal plant samples revealed
aflatoxin B1 in 17 samples at an average of from 10 to 160 mg/kg, ochratoxin A in three samples at
an average of from 20 to 80 mg/kg, and no detection of penicillic acid, zearalenone or Ttoxin.Karan et al. (2005) found ochratoxin A in concentration range of 26–33 μg/kg in allspice,
oregano and hot pepper but did not prove the presence of aflatoxin B1 and G1. Beside aflatoxin
detected in 16 samples of black pepper, fennel, caraway, marjoram, dill and allspice in
concentration range 8–35 μg/kg, El-Kady et al. (1995) also found 10–23 μg/kg sterigmatocystin in
ten samples of paprika, caraway and marjoram.
A literature review on the incidence of mycotoxins as contaminants of various seasonings
indicated the presence of aflatoxins (Vrabcheva, 2000), which are more frequently found in red
peppers (paprika, chilly and capsicum), nutmeg, mustard and ginger. High concentrations of
aflatoxins are frequently detected in nutmeg, particularly, aflatoxins B1 and B2. Freire et al.
(2000) isolated a wide range of field and storage fungi, totaling 42 species from black pepper, white
pepper and Brazil nut. Chaetocin, penitrem A and xanthocillin were identified only from black
pepper, while tenuazonic acid was identified from both black and white pepper. Aflatoxin G2,
chaetoglobosin C and spinulosin were identified from poor quality Brazil nuts. Leistner and Pitt
(1977) found that, out of 442 Penicillium isolates, 44 synthesized penicillic acid, 17 ochratoxin A,
11 penitrem, 10 citrinin, 6 patulin and 3 produced both patulin and citrinin. Overy and Frisvad
(2005) studied the mycotoxin production and post-harvest storage rot of ginger
(Zingiberofficinale). He found that P. brevicompactum to be the predominant species isolated from
85% of the samples. Mycophenolic acid was identified from corresponding tissue extracts.
Aflatoxin production at various stages of development in cardamom and black pepper was
reported by Banerjee et al. (1993). The toxin was assessed using MAB-based ELISA.
All A. flavus isolates tested produced aflatoxin B1 in amounts ranging from 65 to 3000 ng/ml. In
another study by Geetha and Reddy (1990), A. flavus was indicated in the production of
carcinogenic aflatoxin, mainly in ginger, mustard, garlic and pepper. The highest fungal counts
were observed in black pepper and the lowest in curry leaves. When three spices – corianders,
fennel and ginger collected from Bihar were screened for aflatoxin producing
fungi, A. flavuspredominated and most isolates produced only aflatoxin B1 in varying amounts
(Prasad et al., 1984). Cooking experiments showed that aflatoxin levels in spiced sauces are not
reduced by domestic cooking with either microwave or conventional gas oven heating (Macdonald
and Castle, 1996). Klieber (2001)studied the aflatoxin contamination level in various chilly
9
products like chilly powder, paprika spices, dried fruit and sauces in retail stores in Adelaide,
Australia and its management.
Our results revealed that, the fifteen investigated spices could be grouped into three categories
based on their affinity to be contaminated with moulds (Fig. 1). The first groups include spices
which produced > 1000 cfu/g and were considered to have a high affinity to contamination. This
group included ginger, fenugreek, fennel, garden thyme, red pepper, sweet cumin and aniseed. The
highest contaminated spice was ginger (5325–6800 cfu/g) (Fig. 2), however, the other six spices
were relatively low contaminated with fungi (1010–2300 cfu/g). The second group was
moderately contaminated with fungi (100–1000 cfu/g). Cinnamon, green cumin, pepper, green
cardamom, caraway and cloves were belonged to this category. Cloves were the lowest
contaminated spice in this group and had 163–300 cfu/g (Fig. 3). This could be due to antimicrobial
properties of cloves and its essential oils that are highly effective against moulds (Neilsen and Rios,
2000; Guyenot et al., 2003). The third group was considered as very low contaminated spices and
produced <100 cfu/g (Fig. 4). Sumac was the only spice contained in this group and had 50–63
100 cfu/g. Incidence of various mycobiota on the examined spice samples appear to be comparable
to or lower than those reported on spices from other countries (Aziz et al., 1998; Elshafie et al.,
2002). The present study proves antifungal effect of sumac and its low affinity to be contaminated
with moulds. The antimicrobial activity of sumac extract against the Gram-positive microorganisms
was reported by other investigators (Nasar-Abbasa and Kadir Halkman, 2004).
From the table1, Enteric bacteria viz Enterobacter amnigenus 2, E. agglomerans 1, E. cloacae, E.
sakazakii and other gram negativ e
bacteria
viz
Acinobacter sp.
Aeromonas
salmonicida, Chryseomonas luteola, Flavobacterium sp. Chromobacterium violaceum, Pseudomonas
aeroginosa, P . putida and P .
fluorescens were encountered. Aniseed harboured the
highest number of bacterial load, while the least was encountered on royco shrimp cube and royco
beef cube.
Bacterial species, including five members of Enterobacteriaceae, Enterobacter agglomerans, E.
cloacae, and E. sakazakii were isolated in (aniseed and rosemary), Serratia plymuthica (in maggie
onion cube and maggie shrimp cube) whereas Aeromonas salmonicida and Pseudomonas sp. were
encountered in all the samples. The other species of bacteria were Flavobacterium (aniseed and
rosemary), Chromobacterium violaceum (aniseed), Pseudomonas putila (aniseed and rosemary), P.
aeroginosa (aniseed), Acinobacter sp. (royco shrimp and maggie onion) and P. cepacia (maggie
onion, royco shrimp and royco beef). Previous studies by (Addo, (2005) have shown that similar
microorganisms contaminate Ghanaian spices. Bacterial species, belonging to the family
Enterobacteriaceae frequently causes diarrhea and gastrointestinal infection accounting for an
annual mortality rate of five million people worldwide (Brooks et al., 2001) It is the second most
common cause of death after cardiovascular illness (Talaro and Talaro, 1993).
From Table 2, it is observed that that cinnamon had the lowest percentage (33%) of positive
growth of C. sakazakii, whereas masala had the highest percentage (67%) of positive growth of C.
sakazakii. In general, 50% of all the spices sampled were positive for C. sakazakii. Cinnamon may
have a greater level of antimicrobial properties as compared to the other spices .
From table 3, show that desiccation at 58°C for 50 min and then storage in refrigeration
temperature was able to eradicate all C. sakazakii ATCC 29544 as there was no growth when it
was analyzed after all the different time intervals which were after 30 min, 24, 48 and 72 h.
However, it was not the case with the black pepper that was exposed to desiccation and then
10
stored in room temperature. There was no growth when it was analyzed after 30 min of storage;
however, after 24 h of storage and later time intervals, growth was detected. This signifies that
desiccation treatment may not be effective if the black pepper is stored at room temperature after
being treated .
Surprisingly, flash pasteurization proved to bein effective. Growth of the pathogen was
detected after exposure to pasteurization and storage at room and refrigeration temperatures and
at different time intervals,that is, 30 min, 24, 48 and 72 h. C. sakazakii ATCC29544 was able to
survive the flash pasteurization temperature possibly because it is one of the most thermo
tolerant among the enterobacteriaceae (Nazarowec-White and Farber, 1997).
In all the treatments that were unable to kill the pathogen, it was observed that the pathogen
multiplied as the storage time increased, both in room temperature and refrigeration temperature
and this was because C.
sakazaki ATCC 29544 was able to grow at low temperature (Beuchat et al., 2009). However,
the frequency of growth of the pathogen in refrigeration temperature is expected to differ from
that of the pathogen in room temperature, this was observed in black pepper that was stored in
room temperature which had a heavy growth as compared to the black pepper that was stored in
refrigeration temperature. This is because refrigeration works through the principle of slowing
down the growth of micro organisms (Brackett, 2007).
From table 4, the results of grow out test showed the highest number of symptomatic
seedlings in the variety IISRSreekara (26 %) while in other varieties it ranged from 10-13 %
(Table 4). The major symptom observed in all the varieties was mosaic and no variety specific
symptoms were seen. Other symptoms observed were chlorotic specks, mottling and brittleness of
leaves. When 50 seedlings (including both symptomatic and asymptomatic) of each of the
varieties were subjected to PCR, the highest per centage of PYMoV infected seedlings were seen in
the variety IISR-Sreekara while in other varieties, it ranged from 22-28 (Table 4 ) Compared to the
symptomatological observation, higher percentage of infected seedlings were seen by PCR test
indicating symptomless nature of some of the infected seedlings. The results presented showed
that PYMoV is seed borne in black pepper. The above results showed that PYMoV can be
transmitted through seeds in different varieties of black pepper. A few of the badnaviruses such as
Banana streak virus (BSV) and Dracaena mottle virus (DrMV) are known to integrate their
genome into the host genome. A PCR test can detect both integrated and episomal (virions)
viruses while immunocapture (IC) PCR can detect only episomal viruses . In order to see whether
PYMoV occurs in the form of virions in black pepper berries and seedlings, IC-PCR was carried out.
The results of ICPCR on berries and seedlings using three sets of primer pairs specific to PYMoV
showed positive reaction clearly indicating the presence of PYMoV virions in berries and
seedlings. The identity of the amplicon was confirmed through sequencing and comparison with
PYMoV sequences from GenBank. These results clearly confirm the existence of true seed
transmission of PYMoV in black pepper and hence, seeds can also serve as primary source of
inoculum for the virus. Seed transmission was reported in a few Badnavirus species like KTSV
(upto 40 %) , ComYMV (upto 11 %) and CSSV . Different varieties of the same host species and
even among
different plants in a variety often vary widely in the rate at which seed transmission of a particular
virus occurs In the present study, the variety IISRSreekara showed the highest percentage of
infection both based on symptoms and PCR tests. Earlier studies reported non-transmission of
PYMoV through black pepper seeds based on visual observation of symptoms and immunosorbent
electron microscopy (ISEM) tests on seedlings raised from berries collected from infected black
pepper plants. But results of the present work clearly demonstrated the occurrence of true seed
11
transmission of PYMoV in black pepper based on symptoms and PCR tests. This is the first report of
occurrence of seed transmission of PYMoV. Seed transmission may not have much significance in
the spread of PYMoV in black pepper plantations as black pepper is mainly propagated through
vegetative means.
In breeding experiments this can cause considerable interference. The seed can be infected when
one of the parents is infected as there is chance for the pollens and ovary to be infected. The
present study also demonstrated the development and utility of IC-PCR in detecting PYMoV virions
in plants and berries.
References:
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Hashem, Mohamed, and Saad Alamri. "Contamination of Common Spices in Saudi Arabia
Markets with Potential Mycotoxin-producing Fungi." Saudi Journal of Biological Sciences 17.2
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spice products in Ghana” African Journal of Environmental Science and Technology 15.8(2011).
Madela Nokwanda and Oluwatosin A. Ijabadeniyi*, Food borne bacteria isolated from spices
and fate of Cronobacter sakazakii ATCC 29544 in black pepper exposed to drying and various
temperature conditions , Department of Biotechnology and Food Technology, Durban
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Molecules." Open Access. N.p., n.d. Web. 26 June 2012.
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pag. Web. Mar. 2012.
P.S. Hareesh and A.I. Bhat. “Seed transmission of Piper yellow mottle virus in black pepper (Piper
nigrum L)” Journal of Plantation Crops 28.12.(2009)
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