ORIGINAL RESEARCH

published: 25 May 2020

doi: 10.3389/fpls.2020.00670

Potato Cyst Nematodes: A New

Threat to Potato Production in East
Africa
Harrison Mburu 1,2,3 , Laura Cortada 2,4 , Solveig Haukeland 1,5 , Wilson Ronno 6 ,
Moses Nyongesa 7 , Zachary Kinyua 7 , Joel L. Bargul 1,3 and Danny Coyne 2,4*
1
International Centre of Insect Physiology and Ecology (icipe), Nairobi, Kenya, 2 East Africa Hub, International Institute of
Tropical Agriculture (IITA), Nairobi, Kenya, 3 Department of Biochemistry, Jomo Kenyatta University of Agriculture
and Technology, Juja, Kenya, 4 Nematology Section, Department of Biology, Ghent University, Ghent, Belgium,
5
Biotechnology and Plant Health Division, Norwegian Institute for Bioeconomy Research, Ås, Norway, 6 Food and Agriculture
Organization (FAO), Nairobi, Kenya, 7 Kenya Agricultural and Livestock Research Organization (KALRO), Nairobi, Kenya

Potato cyst nematodes (PCN), such as Globodera rostochiensis and Globodera pallida,
are quarantine restricted pests of potato causing major yield and financial losses to
farmers. G. rostochiensis was first reported from Kenya’s key potato growing area
in 2015. We sought to determine the diversity, prevalence and distribution of PCN
Edited by:
species across the country by conducting a country-wide survey between 2016 and
Christina Cowger, 2018, which included a more focused, follow-up assessment in three key potato
Plant Science Research Unit
growing counties. A total of 1,348 soil samples were collected from 20 potato growing
(USDA-ARS), United States
counties. Information regarding local potato farming practices, potato cultivar use, their
Reviewed by:
Josselin Montarry, diversity and availability was also recorded. PCN cysts were obtained from 968 samples
INRA Centre Bretagne-Normandie, (71.8%) in all the counties surveyed, with Nyandarua County recording the highest PCN
France
Louise-Marie Dandurand,
field-incidence at 47.6%. The majority of PCN populations, 99.9%, were identified as
University of Idaho, United States G. rostochiensis, while G. pallida was recovered from just one field, in a mixed population
*Correspondence: with G. rostochiensis. Inconsistencies in PCR amplification efficiency was observed for
Danny Coyne
G. rostochiensis using the recommended EPPO primers, compared with ITS primers
D.Coyne@cgiar.org
AB28/TW81, indicating that this protocol cannot be entirely relied upon to effectively
Specialty section: detect PCN. Egg density in Nyandarua County varied between 30.6 and 158.5 viable
This article was submitted to
eggs/g soil, with an average egg viability of 78.9 ± 2.8% (min = 11.6%, max = 99.9%).
Plant Microbe Interactions,
a section of the journal The PCN-susceptible potato cultivar named Shangi was the most preferred and used by
Frontiers in Plant Science 65% of farmers due to its shorter dormancy and cooking time, while imported cultivars
Received: 30 January 2020 (Destiny, Jelly, Manitou, and Markies) with resistance to G. rostochiensis were used
Accepted: 29 April 2020
Published: 25 May 2020
by 7.5% of farmers due to unavailability and/or limited access to seeds. Thus, most
Citation:
farmers preferred using their own farm-saved seeds as opposed to purchasing certified
Mburu H, Cortada L, seeds. Establishing the distribution and prevalence of PCN and elucidating the local
Haukeland S, Ronno W, Nyongesa M,
farming practices that could promote the spread of PCN is a necessary precursor to
Kinyua Z, Bargul JL and Coyne D
(2020) Potato Cyst Nematodes: the implementation of any containment or management strategy in the country and
A New Threat to Potato Production ultimately across the region.
in East Africa.
Front. Plant Sci. 11:670. Keywords: EPPO, Globodera rostochiensis, Globodera pallida, Kenya, smallholder farmers, potato seed systems,
doi: 10.3389/fpls.2020.00670 cv. Shangi

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Mburu et al. PCN Threat in East Africa

INTRODUCTION after the appearance in the rhizosphere of specific root diffusates

from a narrow range of hosts, mainly potato, although other
Potato is a valuable and nutritious staple crop, driving both solanaceous crops, such as tomato, eggplant or pepper can also
food security and Growth Domestic Product (GDP) growth stimulate egg hatch (Perry, 2002). The infective juveniles (J2)
globally (Reader, 2009; Devaux et al., 2014). Approximately half readily hatch in the presence of a host plant, yet in the absence
of the world’s potato is produced in Asia, especially China, of a suitable natural host cyst decline has been reported to be
followed by Europe producing about a third (Scott and Suarez, ∼0–20% in temperate regions, year-on-year (Devine et al., 1999).
2012). Only maize is grown in more countries than potato. PCN decline under subtropical or tropical conditions in Africa
Africa produces about 7% of global potato output, mainly in is currently unknown. Yield losses associated with PCN will vary
Egypt and South Africa (Devaux et al., 2014). Potato production according to conditions, and earlier estimates in the EU suggest
in the East and Central Africa highlands offers great promise that for every 20 viable eggs/g soil ∼2.75 t/ha of potatoes are
despite substantial fluctuations in yields recorded over the last lost (Brown and Sykes, 1983). PCN is mainly spread through
two decades (FAOSTAT, 2018). Potatoes are a popular and cyst-contaminated soil that adheres to the farm machinery,
valuable crop for both food security and income generation, equipment or seeds.
competing well with maize in the subtropical climates at higher The occurrence of PCN presents a key threat to potato
altitudes. Under these conditions, year-round production can be production in Kenya, as well as to the entire East Africa
possible, often with at least two seasons per annum. In recent region where potato features prominently as a food security
years, however, yields have shown notable declining trends, crop or for income generation for millions of smallholder
mainly attributed to major disease outbreaks, inappropriate farmers (VIB, 2019). In order to mitigate the PCN threat in
cropping practices by farmers, substandard seed quality and potato production, the establishment of the level of infestation,
lack of organized market infrastructure for produce (Janssens their geographical distribution, and the agronomic and social
et al., 2013; Kaguongo et al., 2014). Emerging markets for factors that could be influencing their distribution and spread
processed potatoes (e.g., chips, crisps, starch) furthermore, have are essential toward establishing and implementing a national
increasingly focused attention on potatoes, with rising demand management strategy. This may include exploring alternative
from the fast food industry and processing for added economic sampling strategies, which would contribute to a detailed
value (Abong and Kabira, 2013). Processed potatoes, however, understanding of the spread of PCNs (Goeminne et al., 2011,
also demand high levels of quality, which can be difficult to 2015; Mimee et al., 2019). Consequently, this study presents the
sustain in the face of high pest and disease pressures (Janssens results of a countrywide survey, undertaken to determine the
et al., 2013; Kaguongo et al., 2014). Thus, any action to improve distribution of PCN and the potential damage it is causing in
potato production will have a considerable impact on food the major potato growing regions of Kenya. We additionally
security and income in these countries (Scott et al., 2013; examined farmer potato production practices and how these
Haverkort and Struik, 2015; FAO, 2017b; Okello et al., 2017; will need to be taken into consideration for the implementation
Harahagazwe et al., 2018). of future pest management strategies. The information we
In Kenya, potato is the second most important staple food provide here should further help policy makers and regional
crop after maize and valued at ∼$500 million USD annually stakeholders to make informed decisions related to PCN
(CIP, 2019). About 800,000 Kenyans directly benefit from potato containment and mitigation.
production, while across the whole value chain about 2.5 million
people receive income from potato (Abong and Kabira, 2013).
However, in Kenya, yields have declined and currently average MATERIALS AND METHODS
9–10 t/ha, much below the potential of 20–40 t/ha (Kiptoo et al.,
2016; VIB, 2019), and as reflected across the region attributed to Study Sites and Sampling Strategy
factors listed above. The situation is not helped by the emergence In an initial national survey, potato fields in 20 potato
of new pests and diseases, such as the recently detected potato growing counties in Kenya were surveyed between 2016 and
cyst nematodes (PCNs) Globodera rostochiensis and G. pallida 2017, selecting approximately 1% of the potato production
(Mwangi et al., 2015; Mburu et al., 2018). PCNs are subject area per county, in line with the EU directive 2007/33/EC
to strict quarantine regulations in over 100 countries (EPPO, (Official Journal of European Union, 2007). A follow-up in-
2017) and are globally considered as the most important pests depth sampling was undertaken in 2018 in the major potato
threatening potato production but are all too often overlooked in producing counties of Elgeyo Marakwet and Nyandarua as
less developed countries (Coyne et al., 2018; Niere and Karuri, well as Taita Taveta, which presents significant potential for
2018). The quarantine status of PCNs is, in part, related to potato production (Jaetzold and Schmidt, 1983). A focused,
their ability to produce quiescent structures known as cysts in-depth study was also undertaken in Nyandarua County in
that consist of the hardened body of the females measuring order to estimate yield losses being experienced from PCN.
∼0.5 mm in diameter enclosing ∼300–500 eggs each. Cysts Mean annual temperatures in surveyed counties ranged between
persist in the soil for long periods of up to 20 years, even in 12.9 and 35◦ C, although lower temperatures of 2◦ C was
the absence of an appropriate host and can withstand extreme recorded in some areas in Nyandarua County where occasional
cold temperatures (−15◦ C) and prolonged desiccation periods frosting can be experienced. For each sampled field the GPS
(Folkertsma et al., 1997). This diapause cyst stage is broken coordinates were recorded, and a semi-structured questionnaire

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Mburu et al. PCN Threat in East Africa

administered to capture farmer potato production practices and size of the amplicons against a 100 bp ladder (New England
(Supplementary Material). A composite soil sample of ∼1.5 kg Biolabs , Inc., United States). For sequencing, species-specific
R

comprising of 50 sub-samples (cores) was randomly collected PCR reactions were conducted using a singleplex approach with
from each field using a hand trowel from the top 30 cm ITS5/PITSp4 primers for G. pallida and ITS5/PITSr3 for G.
of soil following a “zigzag” pattern (Coyne et al., 2018), and rostochiensis; PCR-amplicons were purified using the QIAquick
placed in a plastic container. When targeting certified potato PCR Purification Kit (Qiagen, United States) and sequenced by
seed farms (22 sites), ∼2.0 kg of composite soil samples Sanger sequencing. DNA sequences were manually edited using
were collected using the same sampling pattern as described BioEdit Sequence Alignment Editor (Hall, 1999). The edited
above. Each sample was well secured, labeled, placed in a sequences were analyzed using NCBI-BLAST tool and compared
cooler-box and transported to icipe laboratories in Nairobi, with previously isolated Kenyan isolates (Mwangi et al., 2015)
Kenya, for processing. and other Globodera spp. to generate a phylogenetic tree using
Seaview software version 4.5.0 (Gouy et al., 2010).
Isolation and Identification of Potato
Cyst Nematodes Incidence, Infestation and Yield Loss
The soil samples were transported to the Kenya Plant Assessment
Health Inspectorate Services (KEPHIS), where they were A focused, more in-depth study was undertaken in Nyandarua
air-dried, thoroughly mixed, sieved to remove stones and County, which has the largest area of cultivated potato in Kenya.
debris before extraction of cysts from a 200 g sub-sample Data related to incidence and infestation level of PCN across
using the Fenwick can floatation method (EPPO, 2013b). surveyed farms were collected from five sub-counties (Kinangop,
Briefly, using a moderate but constant flow of water, each Kipipiri, Ol’Kalou, Ol’Joro Orok and Ndaragua). For each sub-
sample was washed through a 1 mm aperture sieve into county, the average PCN-infestation level was calculated as the
the Fenwick can. Organic matter that passed through the mean number of cysts per 200 g soil recovered from all the
840 µm sieve in the can was collected from the overflow farms visited. Incidence (%) at the county level was further
onto a 250 µm sieve. The sieve was backwashed and the determined as the number of fields where cysts were isolated
final filtrate containing the cysts collected into 200 ml plastic from the total number of sampled farms and expressed as a
beakers. Extractions were then collected on milk filter papers percentage. Based on these results, counties were classified as
and air-dried. The cysts were individually handpicked using low PCN-incidence (50% ≤ X < 70%), mid PCN-incidence
entomological forceps and counted using a Leica MZ12.5 (70% ≤ X < 90%) and high PCN-incidence (X ≥ 90%). Further
dissection microscope. Cysts were placed in a 1 ml Eppendorf classification for the in-depth study in Nyandarua County was
tube and stored at 4◦ C; the remaining soil was stored in undertaken at the sub-county level, where infestation levels were
case of further use. classified according to: 1 < X < 25 cysts, 26 < X < 110 cysts,
Cysts recovered from samples were morphologically 111 < X < 230 cysts, 231 < X < 495 cysts, and 496 < X < 985
identified, based on the EPPO (2017) taxonomic guide, under cysts per 200 g of soil.
a Leica MZ12.5 dissection microscope. The number of cysts The impact of PCN on potato production was estimated using
(empty or containing eggs) positively identified as PCN were the Brown and Sykes (1983) regression line, which estimates that
counted and recorded. About 1–10 cysts recovered from samples for every 20 viable eggs/g soil 2.75 t/ha of potato are lost to PCN.
were subjected to molecular identification using modified EPPO Therefore, PCN egg viability (EV) was assessed using a modified
multiplex-PCR protocols (EPPO, 2017; Mburu et al., 2018) and protocol, adapted from Faggian et al. (2012), as described below.
ITS primers (Joyce et al., 1994) for species identification. DNA Cyst infestation levels varied across the fields within the county,
amplification was carried out using ProFlex PCR systemsTM as was the number of fields with different levels of infestation.
Base thermocycler (Applied Biosystems Life Technologies) In order to assess a proportionate number of cysts from fields
with multiplex primers (PITSr3: 5’-AGC GCA GAC ATG CCG with different infestation levels, fields were grouped according
CAA-3’, PITSp4: 5’-ACA ACA GCA ATC GTC GAG-3’and to infestation level and then cysts were collected from the fields
ITS5 5’-GGA AGT AAA AGT CGT AAC AAG G-3’), which within each cluster group (minimum 10 and a maximum 50 cysts
are species-specific for G. pallida (265 bp amplicon) and/or per field). The fields which had over 20 cysts per 200 g soil were
G. rostochiensis (434 bp amplicon) targeting the 18S rRNA grouped into four clusters and a total of 46 fields were included in
gene and the internal transcribed spacer ITS1 region and ITS the assessment: cluster 1 [fields with 200 g soil (n = 14)], cluster
primers (TW81 5’-GTT TCC GTA GGT GAA CCT GC-3’ and 2 (between 100 and 200 cysts (n = 11)], cluster 3 (between 50
AB28 5’-ATA TGC TTA AGT TCA GCG GGT-3’) targeting the and 100 cysts (n = 5)], and cluster 4 (between 20 and 50 cysts
ITS1-5.8S-ITS2 regions (1,100 bp amplicon). Each PCR reaction (n = 16)]. The samples were assessed for egg viability using Nile
mixture (One Taq 2X Master Mix with standard buffer, New
R
Blue stain, which stains dead eggs and/or juveniles (J2) (Ogiga
England Biolabs Inc., United States) contained 12.5 µl PCR
R
and Estey, 1974), allowing the visual differentiation between dead
master mix (One Taq 2X Master Mix with standard buffer),
R
(non-viable) and live (viable) J2s and eggs (non-stained) under
1 µl of each primer (forward and reverse), 8.5 µl nuclease-free the microscope. For the viability tests, cysts were handpicked
water and 2 µl of DNA (template) totaling 25 µl volume per and placed inside a modified 1.5 ml Eppendorf tube; the end of
reaction. PCR amplicons were electrophoresed through 2% the tube was cut-off and a nylon mesh glued across the bottom.
agarose gel at 100V for 1 h to confirm successful amplification The tubes were placed inside a 24-well flat-bottomed culture

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Mburu et al. PCN Threat in East Africa

plate (Falcon , Thomas Scientific) with 1 ml of 0.01% Nile Blue

R
RESULTS
stain and incubated in the dark at room temperature for 48 h.
After incubation, the stain was carefully rinsed and replaced Incidence, Distribution and Identification
with 1 ml sterile distilled water. Cysts were then individually
of Potato Cyst Nematodes
removed, placed on a glass slide and gently cut open to expose
Cysts were detected from farms in all the counties prospected
the contents (Faggian et al., 2012). The total number of J2s, viable
and were recovered from a total of 902 field samples (82.8%)
and non-viable eggs were counted to determine cyst fertility
during the initial survey (2016–2017) and in 66 fields (25.6%)
(CF). Subsequently, cyst viability (CV%) was calculated as the
in the follow-up survey in 2018. Nyeri County presented the
total number of live J2s and viable eggs divided by the CF and
lowest incidence (53%), while counties such as Trans Nzoia, Taita
expressed as a percentage (CV%).
Taveta and West Pokot had 100% incidence (Figure 1). Of the
Finally, in each field, soil infectivity (SI) was calculated as the
22 certified seed farms sampled, 82% (n = 18/22) farms were
mean number of viable eggs per cyst, multiplied by the total
infested with PCN.
number of cysts extracted in 200 g soil and expressed as viable
eggs/g soil. For each sub-county, the mean SI was calculated
Detection and Molecular Identification of
from all the studied fields. In each sub-county, potato yield losses
(t/ha) directly attributable to PCN were estimated based on the SI Potato Cyst Nematodes
damage threshold determined by Brown and Sykes (1983). Of 968 samples that had sufficient numbers of cysts to
enable molecular diagnosis, 170 (17.6%) were identified as
G. rostochiensis (GenBank accession: MN378644.1, MN378550.1,
Detection of Potato Cyst Nematode
MN378566.1, 382342.1 – MN382349.1) from a combination of
Resistance H1 Gene both protocols. A relatively lower PCR amplification efficiency
Molecular analyses was conducted on the most popular potato was observed with EPPO primers in PCN-species identification.
cultivar grown in Kenya, cv. Shangi (Sinelle, 2018) and four However, cysts from 61 fields were amplified using ITS primers
cultivars recently introduced from the EU (cv.s Destiny, Manitou, AB28/TW81 (Joyce et al., 1994), which produced amplicons
Markies, and Jelly) (NPCK, 2019) to determine the presence of in samples that previously did not yield any PCR products
the H1 gene, which confers resistance to G. rostochiensis. The using the same DNA template with the EPPO primers. Samples
cv. Jelly, which is resistant to G. rostochiensis with H1 gene amplifying with EPPO primers (ITS5/PITSr3) were recovered
(NPCK, 2017), was used as a positive control and cv. Desiree from 6 counties, 144 in Nyandarua (46.6%), 13 in Narok (20.3%),
as the susceptible control. DNA was extracted from three leaves 6 in Kiambu (7.0%), 3 in Nakuru (2.2%), 3 in Taita Taveta (27.3%)
of 1-month old potato plants grown in the greenhouse at icipe, and 1 in Nyeri (2.2%). PCR products obtained using ITS5/PITSr3
Nairobi. PCR amplification of the genomic region containing primers were sequenced (MN382342 to MN382345 Kiambu
H1 gene was conducted using 57R, TG689 and BCH markers County, MN382346 and MN382347 Nyandarua County, and
(Park et al., 2018). MN382348 and MN382349 Narok County) and analyzed using
the NCBI-BLAST tool. Our findings show high sequence identity
Determining Farmer Practices and (%) ranging from 90 to 95% to G. rostochiensis populations from
Preferences in Potato Production EU, Asia, and Northern and Southern Africa, including those
To understand how current smallholder farming practices could previously described in Kenya (Figure 2).
be influencing the incidence and distribution of PCN, farmer
interviews were conducted at the household level (HH) during Detection of the H1 Resistance Gene
field sampling. A semi-structured questionnaire was used to The molecular analyses of potato cultivars confirmed the
gather agronomic and socioeconomic data to complement the presence of the H1 gene in cv.s Manitou, Markies, Destiny and
phytosanitary data obtained from the epidemiological survey. Jelly (positive control) but not for cv. Shangi, indicating its
Data on farm size (acres), potato cultivars grown, potato seed susceptibility to G. rostochiensis (Table 1).
source, and crop management strategies, e.g., rotation periods,
use of alternate crops, etc. were collected. Enumerators were Potato Cyst Nematode Infestation and
trained prior to conducting the interviews. Data were collected Yield Loss Assessment
directly onto tablets and collated using Epicollect 1 software.
R
The potential impact of PCN on potato yield from the in-
depth survey of 86 fields in Nyandarua County showed that
Data Analysis PCN were recovered from 72.1% of sampled fields and prevalent
The data was analyzed using Microsoft Excel Version 1911,
R R
in all sub-counties. The highest incidence was observed in
mean and standard error of the means were calculated using the Kinangop (92.9%), followed by Kipipiri (80.0%), Ol’Joro Orok
“psych” package, while the model of significance was detected (78.6%), Ol’Kalou (52.9%) and Ndaragua (33.3%). Among these,
by analysis of variance with a chi-square test and the pairwise infestation levels varied: between 1 < X < 25 cysts (24.4%),
comparison of the treatments undertaken using Tukey’s multiple 26 < X < 110 cysts (22.1%), 111 < X < 230 cysts (14.0%),
comparison test using the “lsmeans” on R version 3.6.1. 231 < X < 495 cysts (5.8%), and 496 < X < 985 cysts
(5.8%) respectively per 200 g soil. On average, higher infestation
1
http://www.epicollect.net/ levels were found in Kinangop, with the largest number (66.7%)

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Mburu et al. PCN Threat in East Africa

FIGURE 1 | PCN distribution and incidence level classification across the 20 counties in Kenya.

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Mburu et al. PCN Threat in East Africa

FIGURE 2 | Phylogenetic relation of Kenyan PCN populations compared to those reported in Africa and rest of the world.

TABLE 1 | Detection of Globodera rostochiensis (Ro1) H1 resistance gene in cultivars across Kenya (Table 3), among which cv. Shangi was
Kenyan grown potato cultivars.
the most preferred (65%, predominant in 13 counties), followed
Cultivars BCH TG689 57R Classification of PCN by cv. Arka (10%, predominant in Bungoma and Trans Nzoia
primers primers primers resistance1 Counties). Based on interviews, the overwhelming preference
of cv. Shangi is attributed to its short dormancy (∼5–6 weeks)
Shangi + − − Susceptible (This article)
meaning that it does not require refrigeration/cool storage of
Manitou + + + Resistant
tubers until the following planting season. It also has a fast
Markies + + + Resistant
cooking time, so it requires less fuel and labor for processing (i.e.,
Destiny + + + Resistant
boiling). This makes it particularly appealing to women, who are
Desireé + − − Susceptible control
largely responsible for collection of firewood for cooking.
Jelly + + + Resistant control
Most farmers (56.0%; n = 752) identified farm-saved seed,
1 As referenced in the National Potato Council of Kenya NPCK (2019). which is recycled over several seasons, and ware-potatoes bought
from local markets (34.0%; n = 458) as their principal source of
planting material. Government and private institutional sources
of highly infested fields or “hotspots” with cyst counts ≥231 of seed, such as Agricultural Training College-Farms (ATCs)
cyst/200 g soil (Figures 3, 4). (2.3%), seed aid from the Ministry of Agriculture (2.1%), National
The mean CV within Nyandarua County did not differ Research Organizations (KALRO) (1.7%) or private certified
significantly among sub-counties, although mean CF levels were seed multipliers (1.9%) accounted for just 8.9% of seed supply.
greater (p ≤ 0.05) in Kipipiri and Ol’Joro Orok; Kinangop Noteworthy, the diversity on the sources of potato seed used
and Kipipiri presented the highest SI levels (viable eggs/g soil) for planting also varied among counties, although the two
with 158.5 and 90.7 viable eggs/g soil, respectively, while the informal sources for seed (“Farm-saved” and “Markets”) were
lowest SI levels were observed in Ol’Kalou (30.6 viable eggs/g predominant across counties (Figure 5). In counties such as
soil). Yield losses attributed to PCN, considering the damage Bungoma, Kirinyaga, Laikipia, Nyeri, and Trans Nzoia only
threshold of 2.75 t/ha per 20 viable eggs/g soil (Brown and Sykes, “Farm-saved” and “Markets” seeds were acknowledged to be in
1983), ranged from a minimum of 4.2 t/ha in Ol’Kalou to a use. The lowest diversity of potato seed source was observed in
maximum of 21.8 t/ha in Kinangop (Table 2) based on the SI data West Pokot, where “Markets” (ware potato) were the sole source
per sub-county. of seed. The highest diversity of seed supply (n = 7) was recorded
in Nyandarua and Taita Taveta. Only eight counties reported seed
Smallholder Farmer Practices multipliers as an accessible source of planting material, and the
The average household land size dedicated to potato production highest percentage of farmers in Meru County (11.4%) accessed
was 0.35 ha. Farmers acknowledged the use of up to 34 potato quality declared certified seeds (Figure 6). Respondents did not

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Mburu et al. PCN Threat in East Africa

FIGURE 3 | PCN infestation levels across the 5 sub-counties of Nyandarua indicating the “hot-spots” in red.

use certified seeds because of: (i) high cost of potato seeds; (ii) The majority of farmers (89%) indicated that they employed
limited impact experienced in terms of increased yields; (iii) crop rotation to manage potato pests and diseases (Table 4).
scarcity of certified seeds for the most-preferred cultivar (i.e., cv. Thus, in six counties surveyed, 100% of farmers practiced
Shangi) especially at the onset of planting season. rotation, while in the remaining 14 counties crop rotation was

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Mburu et al. PCN Threat in East Africa

FIGURE 4 | A representation of PCN cyst infestation levels across the 5 Sub-counties of Nyandarua County.

TABLE 2 | Potential yield losses calculated from Brown and Sykes (1983) formula using PCN cyst and egg data.

Sub-county Mean number of Mean cyst fertility Mean cyst Mean soil infectivity (viable Estimated mean
cysts* (eggs/cyst)* viability (%) egg/g soil) yield loss (t/ha)

Kinangop (n = 21) 215 ± 64.9 147.3 ± 7.6 77 158.5 21.8

Kipipiri (n = 15) 98 ± 34.9 183.4 ± 13.9 88 90.7 12.5
Ol’Joro Orok (n = 15) 48 ± 21.3 180.6 ± 13.1 82 42.3 5.8
Ol’Kalou (n = 20) 52 ± 31.6 127.6 ± 13.4 79 30.6 4.2
Ndaragua (n = 15) 46 ± 40.0 143.5 ± 22.9 67 37.3 5.1

*Values expressed as mean ± SE; Mean number of cysts recovered from 200 g soil sample.

practiced by 60.0 to 93.1%. The majority of farmers practiced across the region. The recent report of G. rostochiensis in Rwanda
rotations over a period of >1 year (two cropping seasons) to (Niragire et al., 2019) demonstrates that the pest is already present
7 years (14 cropping seasons) after planting the first crop (72.4%), and established elsewhere within the region.
with one exception where farm-saved potato seeds had been During the current study, the inconsistency of positive
cultivated successively in the same field for over 10 years (or at amplifications to diagnose G. rostochiensis between the EPPO
least 20 successive crops) (Figure 7). However, in all rotation protocols (EPPO, 2013a,b) and the Bulman and Marshall (1997)
schemes described by farmers, only 21.2% included a different protocol, indicates that false negatives could occur if only EPPO
crop immediately following the first potato crop. protocols had been used to diagnose Kenyan PCN populations.
The Bulman and Marshall (1997) protocol has been commonly
used in other parts of the world (Vossenberg et al., 2014),
DISCUSSION and specifically in detection of G. rostochiensis and G. pallida
samples from Kenya (Mwangi et al., 2015; Mburu et al., 2018).
Recent studies reported for the first-time the occurrence of Consequently, it is advised that at least two PCR-based protocols
G. rostochiensis in Nyandarua County in Kenya (Mwangi et al., should be used to detect and diagnose PCN populations in the
2015). In the present study, we show that this PCN species region and, importantly, that the EPPO protocol should not be
is widespread and present in particularly high densities across entirely relied upon. However, the presence of other Globodera
the country, thus causing severe yield losses. The PCN species spp. other than PCN, in the country cannot be discounted either
G. pallida, now confirmed in Kenya and sub-Saharan Africa and should also be considered when receiving false positives.
(SSA) from the current study and separately documented as a In the East African region potato is a key commodity both for
new disease report (Mburu et al., 2018), is shown to be currently food security and as a cash crop for household income (Thiele
very restricted in its distribution. However, the country-wide et al., 2011). In Kenya, the crop represents a source of income
distribution of G. rostochiensis, including counties bordering for around 2.5 million people across the value chain (Abong and
Uganda and Tanzania, where potato is also an important staple Kabira, 2013), with between 500,000 to 780,000 people directly
food source, creates great concern in respect to PCN distribution involved in potato farming activities (Janssens et al., 2013). PCNs

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Mburu et al. PCN Threat in East Africa

TABLE 3 | Preferred potato cultivar grown in Kenyan counties and overall South America but have since become established in various
percentage uptake.
potato growing regions around the world (Brodie et al., 1993;
County Cultivar Percentage Overall% Hodda and Cook, 2009). In SSA, excluding South Africa, PCNs
have been detected only in Kenya and Rwanda (Mwangi et al.,
Bungoma Arka 72.4 10 2015; Niragire et al., 2019), with a tenuous report from Sierra
Trans Nzoia Arka 70.0 Leone (EPPO, 2009). The current study, therefore, determines
Taita Taveta Asante 43.6 5 that G. rostochiensis is now firmly established in the region
Bomet Dutch Robjin 50.0 5 and that G. pallida is present. The greater proportion of
Laikipia Humba Thuti 42.9 5 information on the importance of PCN has been established
Embu Kanyoni 30.0 5 from studies in temperate climates. Kenya has a sub-tropical
Kirinyaga Mukura Nooke 78.6 5 climate with an absence of prolonged frosting periods and
Baringo Shangi 75.0 65 relatively mild minimum temperatures across the year. This
Elgeyo Marakwet Shangi 92.6 provides favorable conditions for 2–3 potato cropping seasons
Kericho Shangi 69.6 per year, given enough rainfall or irrigation (Gildemacher et al.,
Kiambu Shangi 71.8 2009). The current study provides the first indication of PCN
Meru Shangi 61.5
damage potential under such agro-ecological conditions, which
Murang’a Shangi 66.7
provides compelling justification that urgent and serious action
Nakuru Shangi 80.9
is needed. Our current study demonstrates that PCN is seriously
Nandi Shangi 77.8
threatening potato production in Kenya, where extremely high
Narok Shangi 89.6
infestation levels are present. Current data indicate that on
Nyandarua Shangi 98.3
average, potato yields are equivalent to 9.9 t/ha, or 24.75%
Nyeri Shangi 57.4
of the potential yield (40 t/ha). This equates to approximately
Uasin Gishu Shangi 92.9
USD $127 million annual potato losses in Kenya, based on
West Pokot Shangi 100.0
two cropping seasons per year of susceptible cultivars, such
as cv. Shangi. Even though the damage threshold used to
calculate production losses in the current study were based
are highly destructive pests of potato, resulting in major losses on data from Europe (Brown and Sykes, 1983; EPPO, 2013a),
to production and incurring significant investment toward their this extrapolation is considered a realistic indication of the
management (Niere and Karuri, 2018). They are indigenous to magnitude of PCN damage to potato production in Kenya. The

FIGURE 5 | Source of planting material across the potato growing counties of Kenya.

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Mburu et al. PCN Threat in East Africa

FIGURE 6 | Diversity of seed sources within each county in all potato growing counties in Kenya.

pervasive presence and high densities of PCN across the country TABLE 4 | Adoption of crop rotation across the 20 surveyed counties in Kenya.
undoubtedly helps to explain some of the current potato yield
County No rotation Rotation Percentage
gaps, and why these have been increasing over recent years.
Given the persistent shortage of certified seed potatoes and Baringo 2 7 77.8
the inherent nature of using farm-saved seed, as highlighted Bomet 3 23 88.5
from the current study, potato production gaps are likely to Bungoma 2 27 93.1
further widen due to low quality of planting material, other pest Elgeyo Marakwet 78 143 64.7
and diseases, or inefficient post-harvest handling and storage Embu 0 9 100.0
conditions (Muthoni et al., 2013). During the current study, PCN Kericho 0 24 100.0
was established in ∼82% of the seed production farms in Kenya. Kiambu 0 123 100.0
These findings challenge the status of the formal seed system Kirinyaga 0 9 100.0
and highlights the urgent need for comprehensive phytosanitary Laikipia 4 6 60.0
control measures to be implemented in commercial potato seed Meru 16 109 87.2
operations (Cortada, 2018). Murang’a 1 8 88.9
Plant parasitic nematodes are, over time, repeatedly Nakuru 13 137 91.3
overlooked as damaging soil-borne pests in Africa (Coyne et al., Nandi 1 9 90.0
2018). Their subterranean habit and often indistinct damage Narok 7 62 89.9
symptoms contribute to this neglect, and lack of awareness. Nyandarua 53 313 85.3
Given that PCN are readily disseminated through the use of Nyeri 9 66 88.0
infected potato seed, the routine use of farm-saved tubers for seed Taita Taveta 0 94 100.0
in Kenya (Abong and Kabira, 2013) and the region (Gildemacher Trans Nzoia 1 8 88.9
et al., 2009) undoubtedly encourages and perpetuates the PCN Uasin Gishu 2 13 86.7
issue. Such inherent lack of awareness across the agricultural West Pokot 0 4 100.0
spectrum has inevitably played its part in facilitating its spread.
How long PCN has been present and how it was introduced
remains open to question. Potato was first introduced to (The Kenya Times, 1985) and for research purposes (McArthur,
Africa in the 17th century by Christian missionaries (VIB, 1989). If we consider the sub-tropical climate, lack of frosting
2019), while evidence of their importation into Kenya dates periods, year-round production with multiple cropping seasons
back over 100 years for agricultural diversification, seed aid per annum of susceptible cultivars, as well as the habitual use of

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Mburu et al. PCN Threat in East Africa

FIGURE 7 | Scheme of potato cropping seasons in 1 year.

farm-saved seed, the introduction of PCN may not have been as farmers considered crop rotations as a suitable cultural practice to
long ago as the high infestation levels would suggest (Nyongesa, control pests and diseases, they were unaware of the importance
2015). The high mean cyst density levels of 98 cysts/200 cc of establishing suitable (non-PCN hosts) and sufficiently long
soil (0.49 cysts/cc soil) and up to 985 cysts/200 cc in Kenya, rotation schemes to manage PCN. Smallholder farmers, who
which equate to 980 million cysts/ha, is way above the official represent 98% of potato growers in Kenya, primarily grow potato
detection threshold of 3.8 million cysts/ha in Europe (Spears, as a cash crop, and for home consumption. They produce
1968; Davie et al., 2017), with mature and female cysts visible to 83% of the national production, with an average farm size
the naked eye attached to roots. The densities encountered are dedicated to this crop of less than 0.4 ha/year (Janssens et al.,
consequently remarkably high and way above European norms. 2013). A lack of suitable land coupled with the prevailing
The agroecological factors in Kenya have therefore quite likely status of potato as an income-generating cash crop deters
supported the “rapid” dispersal of PCN and development of such farmers from practicing fallow or cultivating less lucrative non-
high densities. It is quite possible that the physiological nature host crops. A situation arises therefore with the continued
and hatching behavior of these Kenya populations differ from cultivation of potato in infested farms until potato production
their temperate climate counterparts. For example, the optimum becomes unprofitable. Awareness campaigns are therefore
temperature for hatch of the cereal cyst nematode, Heterodera urgently required to enable farmers to understand the importance
avenae, recovered from Egypt was on average 5◦ C higher than of managing this aggressive pest. As per international regulations
for H. avenae populations recovered from Germany (Baklawa (IPPC, 2008), countries affected by PCN are required to adopt
et al., 2017). This information is important to determine, as strict phytosanitary regulations for seed potato production to
successful management options can be highly dependent on prevent its further spread within and outside their borders. The
such characteristics. results of the current epidemiological study on PCN in Kenya
In line with previous findings (Kaguongo et al., 2014; Sinelle, provide a basis upon which to establish suitable management
2018), farmers interviewed in our study identified cv. Shangi approaches and policies against this destructive nematode pest.
as the most important and preferred potato cultivar. This
farmer-selected cultivar was rapidly and informally introduced
and distributed in the early 2000s (ISSD Africa, 2016) before CONCLUSION
its official release (Sinelle, 2018). Its short dormancy and fast
sprouting nature makes it appealing and a ready source of Our results show that PCN is widely spread across the main
recycled seeds that do not require cold storage between planting potato growing counties of Kenya. Globodera rostochiensis is
seasons. It was quickly adopted by Kenyan farmers, thus replacing widely distributed, while G. pallida is currently highly restricted
other potato cultivars due to increasing market demand, early but present. More robust sampling techniques will be required
maturity and high yielding, despite its susceptibility to pests and to ascertain that fields are free of PCN infestation, rather than
diseases, particularly late blight. To our knowledge, our study having low, below-threshold detection levels. Development of
is the first report confirming the susceptibility of cv. Shangi a suitable tool to validate the estimation of yield losses under
to G. rostochiensis infection. Following the preliminary results Kenyan conditions is urgently required. There is need to create
of our study in 2017 (FAO, 2017a; icipe, 2017), PCN testing awareness with farmers on the importance of using certified
was made a mandatory procedure for seed certification schemes, seed, adoption of PCN resistant cultivars and practicing crop
with resistance considered necessary for potato cultivars (NPCK, rotations. Due to inconsistencies in results from established
2019). Consequently, the identification and introduction of molecular protocols, an in-depth molecular analysis of the
cultivars with similar attributes to cv. Shangi, but with resistance PCN from Kenya would be required to fully characterize and
against PCN could prove highly beneficial to addressing the understand the genetic complexity of cyst nematode populations
PCN problem in Kenya, and the region. In addition, although present, including PCN pathotypes. Given the sub-tropical

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Mburu et al. PCN Threat in East Africa

nature prevailing in potato production areas, it will also be Cluster of the CGIAR. JB was supported by DELTAS Africa
important to establish if this has influenced the phenotypic and Initiative grant # DEL-15-011 to THRiVE-2. The DELTAS
behavioral nature of PCN in Kenya, which will have significant Africa Initiative is an independent funding scheme of the
implications to the development of management options. African Academy of Sciences (AAS)’s Alliance for Accelerating
Excellence in Science in Africa (AESA) and supported by the New
Partnership for Africa’s Development Planning and Coordinating
DATA AVAILABILITY STATEMENT Agency (NEPAD Agency) with funding from the Wellcome Trust
grant # 107742/Z/15/Z and the United Kingdom government.
Reference sequences used in this study for phylogenetic analysis Additional support was obtained from icipe institutional funding
are freely available at the NCBI database (https://www.ncbi.nlm. from the United Kingdom’s Department for International
nih.gov/) under the following accession numbers: MG309873.1, Development (DFID), the Swedish International Development
MG309920.1, MN382342.1 – MN382349.1, MN378550.1, Cooperation Agency (SIDA), the Swiss Agency for Development
MN378566.1, and MN378644.1. and Cooperation (SDC), Federal Democratic Republic of
Ethiopia, and the Kenyan Government. The funders had no
role in the design, data collection, interpretation, or decision
AUTHOR CONTRIBUTIONS to submit this publication.
HM, SH, LC, WR, MN, and ZK designed the study. LC
and HM analyzed the data. DC, SH, MN, LC, JB, and HM
wrote the manuscript. All authors improved and approved the ACKNOWLEDGMENTS
final manuscript.
We acknowledge the Laboratory personnel at KEPHIS and
icipe-IITA nematology lab (NemAfrica) for assistance in the
FUNDING laboratory during sample processing and analysis, Richard
Bett and Benson Mwangi for the help in generating cyst
This study was funded by the Federal Ministry for Economic infestation level maps.
Cooperation and Development through the German Federal
Enterprise for International Cooperation – GIZ Small grant
(2017–2018) Project Number: 16.7860.6-001.00 and FAO SUPPLEMENTARY MATERIAL
(Food and Agricultural Organization of the United Nations)
Technical cooperation project (TCP/KEN/3602). Funding is also The Supplementary Material for this article can be found online
acknowledged from North Carolina State University under Prime at: https://www.frontiersin.org/articles/10.3389/fpls.2020.00670/
Award No. project OPP1118810; Root, Tubers and Bananas full#supplementary-material

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