# Armillaria root rot threatens Cameroon's Penja pepper (Piper nigrum L.)

Sandrine Petchayo Tigang, James Michel Tchotet Tchoumi, Jolanda Roux, Julienne Nguefack, Aime Didier Begoude Boyogueno, Michael Mbenoun, Crescence Virginie Mfegue, Salomon Nyassé, Michel Ndoumbé Nkeng, Gerben Martijn ten Hoopen

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Sandrine Petchayo Tigang, James Michel Tchotet Tchoumi, Jolanda Roux, Julienne Nguefack, Aime Didier Begoude Boyogueno, et al. Armillaria root rot threatens Cameroon's Penja pepper (Piper nigrum L.). Tropical Plant Pathology, 2020, 45 (5), pp.434-543. 10.1007/s40858-020-00361-w. hal-04936057

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# Armillaria root rot threatens Cameroon's Penja pepper (Piper nigrum L.)

Sandrine Petchayo Tigang<fnref n="1" />,<fnref n="2" /> Ⓒ · James Michel Tchotet Tchoumi<fnref n="3" /> · Jolanda Roux<fnref n="4" /> · Julienne Nguefack<fnref n="1" /> · Aime Didier Begoude Boyogueno<fnref n="2" /> · Michael Mbenoun<fnref n="2" /> · Crescence Virginie Mfegue<fnref n="2" /> · Salomon Nyassé<fnref n="2" /> · Michel Ndoumbé Nkeng<fnref n="2" />,<fnref n="5" /> · Gerben Martijn ten Hoopen<fnref n="2" />,<fnref n="6" />

Received: 15 November 2019 / Accepted: 27 April 2020 / Published online: 27 May 2020

© Sociedade Brasileira de Fitopatologia 2020

## Abstract

Penja pepper (Piper nigrum) produced in Cameroon has long been recognized for its exceptional organoleptic quality. The pepper vine is grown using a support tree (Spondias mombin) in Cameroon. A root disease is associated with plant deaths in both plants. The disease symptoms are characterized by collar cracking and gummosis and the disease was tentatively identified as Armillaria root rot. In this work the extent of the problem was characterized by surveying 35 farms in Cameroon. Samples were taken from diseased support trees and pepper vines. Support trees exhibiting typical symptoms were found in approximately one third of the surveyed farms. In these farms, disease incidence and Pepper vine mortality ranged from 1.3 to 50% and 1.2 to 87.7%, respectively. Analysis of the ribosomal internal transcribed spacer region (ITS) locus suggested that the isolates collected from S. mombin and P. nigrum are most likely, which is a first for both hosts, represented by Armillaria camerunensis (Henn.) Volk & Burdsall, a fungus previously associated with declining cacao trees in Cameroon. Given the increasing demand for Penja pepper and the great damaging potential of this root rot, research on management strategies should be prioritized.

**Keywords** Armillaria camerunensis · Disease incidence · Molecular identification · Slow decline · Support tree

## Introduction

The pepper vine (Piper nigrum) is a tropical perennial crop, native to India, which belongs to the Piperaceae family (Ravindran 2000). It is cultivated in several countries including Brazil, Indonesia and Vietnam which are the world's largest producers of pepper (Faostat 2017). According to the International Pepper Community (IPC 2017) pepper is the “King of spices” since it is the most consumed and marketed spice in the world. Besides its popular use in cuisine and seasoning, the spice has shown medicinal attributes. Pepper can help relieve viral respiratory ailments such as flu and colds, as well as digestive problems such as constipation, flatulence and colic (Meghwal and Goswami 2012; Ahmad et al. 2012).

Black pepper was introduced to Cameroon in the 1930s and although the exact origin of first introduction is unknown, the first vines were introduced into Cameroon by the Germans, and later by French Planters and the Cameroon Development Corporation (CDC). They brought in cultivars from Cambodia, the Democratic Republic of Congo (DRC) (at the time the Belgian Congo) and India. It is the descendants

<figure><img src="image_3.png" /></figure>

<footnote>Sandrine Petchayo Tigang
ptsandy2000@gmail.com</footnote>

<footnote n="1">¹ Department of Biochemistry, Faculty of Sciences, University of Yaoundé I, P.O. box 812 Yaoundé, Cameroon</footnote>

<footnote n="2">² Institute for Agricultural Research for Development (IRAD), P.O. box 2067 Yaoundé, Cameroon</footnote>

<footnote n="3">³ Department of Biochemistry, Genetics and Microbiology, Forestry and Agricultural Biotechnology Institute (FABI), Faculty of Natural and Agricultural Sciences, University of Pretoria, Pretoria 0028, South Africa</footnote>

<footnote n="4">⁴ Department of Plant and Soil Sciences, Forestry and Agricultural Biotechnology Institute (FABI), Faculty of Natural and Agricultural Sciences, University of Pretoria, Pretoria 0028, South Africa</footnote>

<footnote n="5">⁵ Centre Africain de Recherches sur Bananiers et Plantains (CARBAP), B.P. 832, DoualaBonanjo, Cameroon</footnote>

<footnote n="6">⁶ CIRAD, UPR Bioagresseurs, St. Augustine, Trinidad and Tobago. Bioagresseurs, Univ. Montpellier, CIRAD, Montpellier, France</footnote>
of this miscellany of cultivars that are now grown in
Cameroon.

The pepper produced in Cameroon has long been recognized
by connoisseurs to be among the world's best, owing to its
outstanding organoleptic attributes. This exceptional quality
has earned Penja black pepper the first sub-Saharan protected
geographical indication (GI) label in September 2013 by the
African Intellectual Property Organization (OAPI), further
boosting its reputation. Although Cameroonian production
remains marginal (<0.02% of global production) (Faostat
2017), it has increased over the past few years by over a factor
of 10, as pepper attracts increasing interest from local farmers,
stimulated by the GI label for Penja pepper and a concomitant
price increase from $4.2 before the GI to $23.6 USD per kg in
2015. Prices oscillated between $20.20 and $23.6 US dollars
between 2016 and 2018.

As is the case for any intensively propagated crop, diseases
and pests are a major constraint limiting black pepper produc-
tivity. Numerous pests and diseases have been reported to af-
fect pepper vines; some of which are well known to cause
severe losses, e.g. Phytophthora foot rot caused by
Phytophthora capsici Leonian, (Ravindran 2000; Truong
et al. 2008), blackberry disease caused by Colletotrichum spe-
cies (Keuh 1990), and slow decline (or yellow disease) which
can result from various pathogens and complexes of biotic and
abiotic stressors (Ramana and Santhosh 2000). In Cameroon,
as cultivation of pepper is intensifying so is the pressure of
phytosanitary problems. In recent years especially, there has
been a growing concern among farmers about the losses asso-
ciated with diseases, which in some cases have resulted in up to
50% mortality in certain pepper plantations. The “Plantations
du Haut Penja” (PHP) organization, the biggest pepper produc-
er in Cameroon, saw its production area reduced from 40 to
21 ha because of disease pressure (Mbodiam 2019). According
to preliminary assessments, these losses are most likely asso-
ciated with a range of biotic and abiotic stressors that remain to
be resolved (Petchayo et al. 2015).

In Cameroon, a disease affects the pepper's support tree, *Spondias mombin* L (hog plum), a native of the Americas and West-Indies. The support tree is critical for pepper cultivation as it provides a structure for optimum growth and productivity of pepper plants (Ravindran 2000). The disease affecting *S. mombin* is characterized by collar cracking, gummosis and the presence of white fungal mycelial fans beneath the bark, which are all typical symptoms of Armillaria root rot (Fig. 1c, d). A preliminary investigation indicated that like the support tree, the pepper vine was also susceptible to this disease (Petchayo et al. 2015). However, further studies are needed to support the development of effective management strategies. The aim of this study was therefore two-fold: 1) to elucidate the cause of the disease by means of DNA sequence analyses and; 2) to assess the incidence of root rot in Penja Pepper farms across the certified geographic area using field surveys.

**Materials and methods**

Field observations and sample collection

The Penja pepper production basin lies within the fourth agro-
ecological zone, as defined by the Institute of Agricultural
Research for Development (IRAD) of Cameroon. This region
straddles the Littoral and South-West administrative regions
of the country. It features a tropical rain forest as native veg-
etation cover and a subequatorial climate type, characterized
by 2500–4000 mm annual rainfall, 22–29 °C mean tempera-
tures and 61–90% relative humidity.

Field observations for root rot and sample collection were
carried out in May–June 2016, on a total of 35 farms, selected
from the database of the association of Penja pepper producers
(Penja Pepper Geographical Indication Group, IGPP for its
abbreviation in French) and based on farmer’s indications
whether the disease was present or not. Farms were distributed
over the districts of Penja-Njombe (n = 14; N4° 38. 799′ E9°
40. 331′), Loum (n = 7; N4° 42. 140′ E9° 46. 318′) in the
Littoral region, and Tombel (n = 13; N4° 39. 685′ E9° 36.
853′) in the South-West region. Each plantation was surveyed
to determine the prevalence of the disease. Two transects,
perpendicular to each other, were laid out in the center of each
pepper field. Each transect was 28 m long and included three
rows of pepper plants planted together with support trees
(S. mombin) at 2 × 2 m spacing. Thus, a maximum of 81
pepper vines and support trees were examined and a sanitary
status was assigned to vines and support trees separately, ei-
ther healthy, diseased, or dead/absent (absence of a support
tree or vine was assumed to be due to the death of the plant).
Potential differences per district and plantation age group (3–
6, 7–10 and > 10 yrs. of age) were analyzed using arcsin-
transformed percentage disease incidence data for support
trees and mortality incidence for vine trees with the appropri-
ate generalized linear model in XLSTAT v. 2019.4.1.

Eleven symptomatic vine/support tree combinations were
randomly sampled in one farm per district. Infected samples
were collected from roots, collar and trunk of pepper and sup-
port trees (Fig. 1a-f) following a purposive stratified random
sampling technique. All samples were placed in paper bags,
duly labeled and brought back to the laboratory for further
analyses. Additional information regarding age of the planta-
tion, associated crops and previous land use was also collected.

Isolation of pathogen

Isolation of suspected *Armillaria* cultures from infected plant
materials was performed as described by Tchotet Tchoumi
et al. (2017). Small pieces (2–3 mm³) of woody tissue were
taken from the advancing edge of lesions and were surface ster-
ilized in 7% sodium hypochlorite for 90 s, then rinsed twice in
sterile distilled water and dried using sterile tissue paper. These
<figure><img src="image_4.png" /><figcaption>**Fig. 1** Disease symptoms in pepper plantations in the Penja region of Cameroon, (a) support trees (S. mombin) most of which are devoid of pepper vines; (b) a dead pepper wine, reminiscent of slow decline; (c) gum exuding from the base of a dying support tree; (d) white mycelial fans underneath the bark of support tree; (e) basal stem cracking of black pepper; (f) white mycelium underneath the root bark of *P. nigrum*; (g) Morphological characteristics *in vitro* of *Armillaria* isolate</figcaption></figure>

sterilized plant materials were plated onto Basidiomycete selec-
tive medium, made up of 2% Malt Extract Agar (MEA) supple-
mented with 2 mg/L benomyl, 2 mg/L dichloran and 0.01 g/L
streptomycin sulphate (Worrall 1991). Plates were incubated at
ambient temperature (25 ± 2 °C) in the dark for two weeks, and
pure cultures were obtained from these by transferring
rhizomorph tips onto new MEA plates, which were also main-
tained at ambient temperature.

DNA extraction, amplifications and phylogenetic
analysis

Mycelium from pure cultures was harvested, freeze-dried and
ground to a fine powder using a cell disruptor machine (Retsch
Gmbh, Germany). Genomic DNA was extracted using the
CTAB protocol as described by Möller et al. (1992). Prior to

amplifications, the initial DNA concentrations were adjusted to
100 ng/µL using a Nanodrop ND-1000 Spectrophotometer
(Thermo Fisher Scientific, Wilmington, DE, USA). PCR am-
plifications targeted the ITS regions (ITS1, ITS2), including
the 5.8S subunit of the ribosomal DNA and was done using the
primer pair ITS1 (5'-TCCGTAGGTGAACCTGCGG-3') and
ITS4 (5'-TCCTCCGCTTATTGATATGC-3') (White et al.
1990). PCR and sequencing reaction mixtures and reaction
conditions were the same as outlined in Tchotet Tchoumi
et al. (2017). DNA sequencing was done at the sequencing
facility of the University of Pretoria. The CLC Main
Workbench v.7.6.1 package was used to edit bidirectional-
sequencing reads and to assemble contigs. The ITS sequences
were subsequently queried against the global fungal DNA se-
quence database in the GenBank nucleotide database using the
NCBI BLASTn search tool (https://blast.ncbi.nlm.nih.gov). A
dataset of *Armillaria* ITS sequences was assembled, comprising representatives from the different localities and hosts (*P. nigrum* and *S. mombin*) targeted by this study, the sequences obtained by Roux et al. (2012) on cacao (*Theobromae cacao*) in Cameroon, as well as all BLASTn hits in GenBank with ≥97% sequence identity. Sequences representing *A. hinnulea* were included in the dataset as outgroups (Table 1). The dataset was submitted to multiple sequence alignment with the online program MAFFT (http://mafft.cbc.jp/alignment/server/) prior to phylogenetic analysis based on Maximum likelihood (ML) criterion using RaxML (Stamatakis 2006) in raxmlGUI 1.3 (Silvestro and Michalak 2012). The analysis used 10 parallel runs and 1000 bootstrap replications and applied the GTR + G model of sequence evolution determined as best fit under the Akaike Information Criterion (AIC) using JModeltest v2.1.6 (Darriba et al. 2012; Miller et al. 2010).

## Pathogenicity test

A pathogenicity test was conducted using six-month old *Piper nigrum* plants obtained from cuttings and kept under natural conditions, and two *Armillaria* isolates (CMW44956 and CMW45278). Two inoculation methods were used: soil inoculation and artificial wounding modified from Ford et al. (2017).

For the soil inoculation technique four mycelial agar plugs of the same size, overgrown with the respective *Armillaria* isolates, were mixed with the soil of each pot containing pepper plants. In the wound inoculation study 5 × 5 mm agar plugs containing three-week-old mycelium from each isolate were inserted into a wound made on the collar of each plant. A total of seven pepper seedlings per fungal isolate were inoculated for each inoculation technique. Seven plants were inoculated with sterile agar plugs to represent the controls. Plants were maintained for four months, with daily watering. At the beginning and over the course of the experiment, several growth parameters were evaluated, such as number of leaves, max plant length and mortality. At the end of the experiment, plants were collected and leaf and root dry weight data were collected. Changes in plant height and leaf area were determined, as well as lesion length, measured from the center of the inoculation point, between the beginning and end of the experiment. The general health status of the roots was also determined. Treatment effects were evaluated by fitting a one-way Anova model in XLSTAT v.2019.4.1.

## Results

### Field observations

The average plantation age of the 35 surveyed farms was around 8 years (3 to 15 years) (Table 2). From a total of

2832 inspected vine/support tree combinations (three support trees were absent), 157 (5.5%) of the support trees were visibly affected and showed symptoms of collar cracking and gummosis. The percentage of affected support trees ranged from zero to more than 50% between farms (Table 2). There was no significant effect of districts (*P* = 0.501). In the Tombel district, 7 out of 13 farms (53.8%) were affected and disease incidence in affected farms ranged from 1.2 to 22.2%. In the Njombe-Penja district 3 out of 13 farms (23.1%) were affected and disease incidence in affected farms ranged from 1.2% to 50.6%. Finally, in the district of Loum 2 out of 9 farms (22.2%) showed symptoms with disease incidences of 3.7% and 45.7%. Some surveyed farms (*n* = 13) had no support trees affected by the disease even though the owners indicated that the disease was present (Table 2). On the surveyed farms for which the farmers had indicated the absence of the disease, the disease was indeed absent. This discrepancy between farmer indications and transect data is partially due to the fact that the transect was laid out in the middle of the plot, where in certain cases indeed no diseased trees were present, although diseased trees were sometimes present outside of the transect area. The transect area was put in the middle of the plot to avoid edge effects for certain other parameters measured.

With regard to the pepper vines, a total of 515 out of 2832 support trees (18.2%) (Table 2) were devoid of vines. In other words, on average a fifth of any plantation was unproductive. Contrary to “*Armillaria*” incidence, all farms had a certain level of pepper vine mortality, however, once again there were no marked differences between the three districts (*P* = 0.123). Pepper vine mortality was often associated with yellowing of the vine before subsequent desiccation. Unfortunately, the etiology of yellowing disease in Penja pepper remains to be resolved. Biotic stressors such as nematodes, *Phytophthora* and *Pythium* species as well as *Fusarium solani* (f. sp. *piperis*) might be involved (Cardoso and Albuquerque 1979; Ramana et al. 1987; Anandaraj et al. 1996; Shahnazi et al. 2012). Although nematodes, *Pythium/Phytopythium* spp. and *Fusarium*, have been found in these pepper plantations (data not shown), it has not been possible to unequivocally link these to the slow decline of pepper vines. Correct identification of these organisms, like for *Armillaria* here, will be a first step. Recent advances allowing the correct determination of e.g. the presence of *Fusarium solani* f. sp. *piperis* (Costa et al. 2017) will be useful in this respect.

For both support tree and pepper vine, disease was not related to plantation age (*P* = 0.208, *P* = 0.958, respectively). This might indicate that the problems/pathogen(s) afflicting support trees and/or pepper vines were already present at the time of plantation establishment or arrived very shortly after.

### Pathogen identification

All sampled diseased plants presented similar symptoms, such as cracking of the bark and wood (Fig. 1c, e) and presence of
<table><caption>**Table 1** Isolates used in the phylogenetic analysis</caption><thead><tr><th>Species</th><th>Strain*</th><th>Host</th><th>Geographical origin</th><th>GenBank accession numbers (ITS)</th><th>Reference</th></tr></thead><tbody><tr><td>Armillaria camerunensis</td><td>TH 9926</td><td>Dead wood</td><td>Cameroon</td><td>KU170951</td><td>Koch et al. 2017</td></tr><tr><td>A. camerunensis</td><td>TH DJA 91</td><td>Gilbertiodendron dewevrei</td><td>Cameroon</td><td>KU170952</td><td>Koch et al. 2017</td></tr><tr><td>Armillaria sp.</td><td>CMW34125</td><td>T. cacao</td><td>Cameroon (B)</td><td>MG254549</td><td>Roux et al. 2012</td></tr><tr><td>Armillaria sp.</td><td>CMW36819</td><td>T. cacao</td><td>Cameroon (N)</td><td>MG254551</td><td>Roux et al. 2012</td></tr><tr><td>Armillaria sp.</td><td>CMW36820</td><td>T. cacao</td><td>Cameroon (N)</td><td>MG254552</td><td>Roux et al. 2012</td></tr><tr><td>Armillaria sp.</td><td>CMW36821</td><td>T. cacao</td><td>Cameroon (K)</td><td>MG254553</td><td>Roux et al. 2012</td></tr><tr><td>Armillaria sp.</td><td>CMW43766</td><td>Spondias mombin</td><td>Cameroon (P)</td><td>MG182003</td><td>This study</td></tr><tr><td>Armillaria sp.</td><td>CMW44921</td><td>S. mombin</td><td>Cameroon (P)</td><td>MG182004</td><td>This study</td></tr><tr><td>Armillaria sp.</td><td>CMW44922</td><td>S. mombin</td><td>Cameroon (P)</td><td>MG182005</td><td>This study</td></tr><tr><td>Armillaria sp.</td><td>CMW44923</td><td>S. mombin</td><td>Cameroon (P)</td><td>MG182006</td><td>This study</td></tr><tr><td>Armillaria sp.</td><td>CMW44924</td><td>S. mombin</td><td>Cameroon (P)</td><td>MG182007</td><td>This study</td></tr><tr><td>Armillaria sp.</td><td>CMW44927</td><td>S. mombin</td><td>Cameroon (L)</td><td>MG182008</td><td>This study</td></tr><tr><td>Armillaria sp.</td><td>CMW44956</td><td>Piper nigrum</td><td>Cameroon (Njombe)</td><td>MG182009</td><td>This study</td></tr><tr><td>Armillaria sp.</td><td>CMW45278</td><td>Piper nigrum</td><td>Cameroon (P)</td><td>MG182010</td><td>This study</td></tr><tr><td>Armillaria sp.</td><td>CMW45281</td><td>S. mombin</td><td>Cameroon (Njombe)</td><td>MG182011</td><td>This study</td></tr><tr><td>A. fuscipes</td><td>CMW10165</td><td>Prunus persica</td><td>Zimbabwe</td><td>AY882966</td><td>Coetzee et al. 2005</td></tr><tr><td>A. fuscipes</td><td>CMW2717</td><td>Pinus elliottii</td><td>South Africa</td><td>AY882971</td><td>Coetzee et al. 2005</td></tr><tr><td>A. fuscipes</td><td>CMW2740</td><td>Pinus patula</td><td>South Africa</td><td>AY882970</td><td>Coetzee et al. 2005</td></tr><tr><td>A. fuscipes</td><td>CMW3164</td><td>Pelargonium asperum</td><td>La Reunion</td><td>AY882975</td><td>Coetzee et al. 2005</td></tr><tr><td>A. fuscipes</td><td>CMW4871</td><td>Widdringtonia whytei</td><td>Malawi</td><td>AY882976</td><td>Coetzee et al. 2005</td></tr><tr><td>A. fuscipes</td><td>CMW4873</td><td>Indigenous shrub</td><td>Malawi</td><td>AY882977</td><td>Coetzee et al. 2005</td></tr><tr><td>A. fuscipes</td><td>CMW4874</td><td>Araucaria cunninghamii</td><td>Zimbabwe</td><td>AY882967</td><td>Coetzee et al. 2005</td></tr><tr><td>A. fuscipes</td><td>CMW4949</td><td>Pinus elliottii</td><td>Tanzania</td><td>AY882978</td><td>Coetzee et al. 2005</td></tr><tr><td>A. fuscipes</td><td>CMW4950</td><td>Pinus strobes</td><td>Tanzania</td><td>AY882979</td><td>Coetzee et al. 2005</td></tr><tr><td>A. fuscipes</td><td>CMW4953</td><td>Pelargonium asperum</td><td>La reunion</td><td>AY882974</td><td>Coetzee et al. 2005</td></tr><tr><td>A. fuscipes</td><td>CMW5844</td><td>Pinus patula</td><td>Ethiopia</td><td>AY882969</td><td>Coetzee et al. 2005</td></tr><tr><td>A. fuscipes</td><td>CMW5846</td><td>Pinus patula</td><td>Ethiopia</td><td>AY882968</td><td>Coetzee et al. 2005</td></tr><tr><td>A. fuscipes</td><td>CMW7184</td><td>Cypress sp.</td><td>Kenya</td><td>AY882973</td><td>Coetzee et al. 2005</td></tr><tr><td>A. fuscipes</td><td>CMW7187</td><td>Camellia sinensis</td><td>Kenya</td><td>AY882972</td><td>Coetzee et al. 2005</td></tr><tr><td>Armillaria sp.</td><td>CMW3173</td><td>Tectona grandis</td><td>Zambia</td><td>AY882981</td><td>Coetzee et al. 2005</td></tr><tr><td>Armillaria sp.</td><td>CMW4455</td><td>Camellia sinensis</td><td>Zimbabwe</td><td>AY882985</td><td>Coetzee et al. 2005</td></tr><tr><td>Armillaria sp.</td><td>CMW4456</td><td>Brachystegia utilis</td><td>Zimbabwe</td><td>AY882984</td><td>Coetzee et al. 2005</td></tr><tr><td>Armillaria sp.</td><td>CMW3152</td><td>Unknown</td><td>Cameroon</td><td>AY882980</td><td>Coetzee et al. 2005</td></tr><tr><td>Armillaria sp.</td><td>CMW10115</td><td>Acacia albida</td><td>Zimbabwe</td><td>AY882983</td><td>Coetzee et al. 2005</td></tr><tr><td>Armillaria sp.</td><td>CMW10116</td><td>Newtonia buchananii</td><td>Zimbabwe</td><td>AY882982</td><td>Coetzee et al. 2005</td></tr><tr><td>A. hinnulea</td><td>NZFS22376</td><td>Unknown</td><td>New Zealand</td><td>EU734746</td><td>Unpublished</td></tr><tr><td>A. hinnulea</td><td>NZFS2394</td><td>Unknown</td><td>New Zealand</td><td>EU734745</td><td>Unpublished</td></tr></tbody></table>

B: Bokito; L: Loum; N: Ngat; P: Penja

*Strain in bold were collected during this study

white fungal mycelial mats beneath the bark (Fig. 1d, e, f). Gum exudation was only observed on support trees (Fig 1c). All isolates collected had the typical culture morphology of *Armillaria* species, such as the presence of abundant rhizomorph growth (Fig. 1g).

PCR amplification and sequencing of isolates yielded ITS amplicons ranging in size from 616 to 618 bp, and showing high (99%) sequence identity with *Armillaria* isolates obtained previously from declining *Theobroma cacao* trees in Cameroon (Roux et al. 2012) and *Tectona grandis* in
<table><caption>**Table 2** Armillaria root rot incidence at three localities in Cameroon</caption><thead><tr><th>District</th><th>Plantation</th><th>Farmer obs.<sup>1</sup></th><th>Armillaria Incidence (%)<sup>2</sup></th><th>Pepper vine Mortality (%)<sup>3</sup></th><th>Age (yrs.)</th><th>Associated crops</th><th>Previous land use</th></tr></thead><tbody><tr><td rowspan="10">Tombel</td><td>T1</td><td>+</td><td>3.4</td><td>27.2</td><td>9</td><td>Prunus africana, Persea americana, Cola acuminata</td><td>Coffea sp.</td></tr><tr><td>T2</td><td>+</td><td>30.0</td><td>87.7</td><td>3</td><td>None</td><td>Coffea sp., T. cacao</td></tr><tr><td>T3</td><td>+</td><td>4.4</td><td>16</td><td>3</td><td>P. africana, P. americana, C. acuminata</td><td>Coffea sp.</td></tr><tr><td>T4</td><td>+</td><td>17.2</td><td>21</td><td>15</td><td>T. cacao, Dioscorea alata</td><td>Coffea sp., A. comosus</td></tr><tr><td>T5</td><td>+</td><td>1.3</td><td>1.2</td><td>7</td><td>T. cacao</td><td>Native forest</td></tr><tr><td>T6</td><td>+</td><td>33.3</td><td>33.3</td><td>15</td><td>Musa sp.</td><td>T. cacao</td></tr><tr><td>T7</td><td>+</td><td>28.6</td><td>56.8</td><td>7</td><td>T. cacao, Musa sp.</td><td>T. cacao</td></tr><tr><td>T8</td><td>—</td><td>—</td><td>21</td><td>10</td><td>T. cacao, D. alata</td><td>None</td></tr><tr><td>T9</td><td>+</td><td>—</td><td>22.2</td><td>5</td><td>Ananas comosus, Musa sp.</td><td>Coffea sp.</td></tr><tr><td>T10</td><td>—</td><td>—</td><td>3.7</td><td>5</td><td>Musa sp.</td><td>None</td></tr><tr><td rowspan="10">Njombe- Penja</td><td>T11</td><td>+</td><td>—</td><td>67.6</td><td>10</td><td>T. cacao, D. alata</td><td>T. cacao</td></tr><tr><td>T12</td><td>—</td><td>—</td><td>19.8</td><td>4</td><td>T. cacao, Musa sp.</td><td>T. cacao, Coffea sp.</td></tr><tr><td>T13</td><td>+</td><td>—</td><td>4.9</td><td>8</td><td>T. cacao, Musa sp.</td><td>Coffea sp.</td></tr><tr><td>N1</td><td>+</td><td>1.3</td><td>3.7</td><td>10</td><td>Citrus sinensis, C. acuminata</td><td>T. cacao</td></tr><tr><td>N2</td><td>+</td><td>5.5</td><td>32.1</td><td>11</td><td>None</td><td>T. cacao</td></tr><tr><td>N3</td><td>+</td><td>51.9</td><td>3.7</td><td>12</td><td>None</td><td>Native forest, Musa sp.</td></tr><tr><td>N4</td><td>+</td><td>—</td><td>6.2</td><td>7</td><td>None</td><td>T. cacao</td></tr><tr><td>N5</td><td>+</td><td>—</td><td>38.3</td><td>8</td><td>Fruit trees</td><td>T. cacao</td></tr><tr><td>N6</td><td>+</td><td>—</td><td>6.2</td><td>25</td><td>Musa sp., D. alata</td><td>C. acuminata, T. cacao</td></tr><tr><td>N7</td><td>+</td><td>—</td><td>9.9</td><td>10</td><td>None</td><td>T. cacao</td></tr><tr><td rowspan="10">Loum</td><td>N8</td><td>+</td><td>—</td><td>18.5</td><td>n.d.</td><td>Musa sp.</td><td>Forest trees</td></tr><tr><td>N9</td><td>—</td><td>—</td><td>8.6</td><td>8</td><td>Musa sp.</td><td>C. papaya, A. comosus</td></tr><tr><td>N10</td><td>+</td><td>—</td><td>32.1</td><td>7</td><td>C. acuminata, Musa sp.</td><td>T. cacao, C. acuminata</td></tr><tr><td>N11</td><td>—</td><td>—</td><td>6.2</td><td>3</td><td>Carica papaya, A. comosus</td><td>Carica papaya</td></tr><tr><td>N12</td><td>+</td><td>—</td><td>2.5</td><td>6</td><td>None</td><td>A. comosus</td></tr><tr><td>N13</td><td>+</td><td>—</td><td>8.6</td><td>6</td><td>Musa sp., C. papaya</td><td>Musa sp.</td></tr><tr><td>L1</td><td>+</td><td>4.5</td><td>18.5</td><td>4</td><td>None</td><td>T. cacao</td></tr><tr><td>L2</td><td>+</td><td>64.9</td><td>28.4</td><td>6</td><td>None</td><td>T. cacao</td></tr><tr><td>L3</td><td>—</td><td>—</td><td>7.4</td><td>16</td><td>C. papaya</td><td>T. cacao</td></tr><tr><td>L4</td><td>+</td><td>—</td><td>4.9</td><td>5</td><td>None</td><td>Coffea sp., Manihot esculenta</td></tr><tr><td rowspan="5">Loum</td><td>L5</td><td>—</td><td>—</td><td>9.8</td><td>10</td><td>Musa sp.</td><td>Zea mays, Musa sp.</td></tr><tr><td>L6</td><td>—</td><td>—</td><td>39.5</td><td>17</td><td>Musa sp., D. alata</td><td>Musa sp.</td></tr><tr><td>L7</td><td>—</td><td>—</td><td>2.5</td><td>8</td><td>None</td><td>T. cacao</td></tr><tr><td>L8</td><td>+</td><td>—</td><td>18.5</td><td>8</td><td>None</td><td>Coffea sp.</td></tr><tr><td>L9</td><td>—</td><td>—</td><td>29.6</td><td>11</td><td>None</td><td>Musa sp.</td></tr></tbody></table>

Zambia (Coetzee et al. 2005). In the ML based phylogram, the *Armillaria* isolates from Penja pepper fields grouped together, with strong bootstrap support (86%), into a single phylogenetic clade, alongside the type *A. camerunensis*, as

well as isolates associated with cacao root rot in Cameroon. This clade also included isolates from diverse host plants in Zambia and Zimbabwe (Fig. 2). The *A. camerunensis* clade is sister to another clade

<footnote n="1">¹ ± presence or absence of the disease in the plantation according to the farmer</footnote>

<footnote n="2">² Armillaria incidence based on transect data, — = absence of Armillaria incidence within the transect</footnote>

<footnote n="3">³ Pepper vine mortality based on transect data</footnote>

<footnote>n.d. = not determined</footnote>
representing *A. fuscipes*, which is well supported by a
strong bootstrap value of 99%.

Pathogenicity test

The pathogenicity test showed no significant differences be-
tween treatments with regard to changes in plant height or leaf
surface area nor for root and leaf dry weight at the end of the
assay (P-values >0.2), although on some uprooted pepper
plants what seemed like necrotic root lesions were observed.
However, a significant difference (P = 0.018, dl = 2, F = 4.97)
was observed with regard to lesion length from the point of
collar inoculation. Control plants had no lesion development,
yet pepper seedlings infected by the pepper isolate had an
average lesion size of 4.5 mm and those infected by the
*S. mombin* isolate on average of 7.1 mm which was signifi-
cantly different from the control. Certain plants presented
yellowing, unfortunately, re-isolation efforts from wounds
and necrotic roots were unsuccessful. Care has to be taken
when interpreting these results. Pepper cuttings take several
months before developing a root system and initially grow
very slow. This, combined with the difficulty of *Armillaria*
pathogenicity assays and the relatively short duration of the
trial (4 months) may be the reason that no effects, other than
the presence of lesions were observed.

Discussion

The ITS based phylogenic tree clearly shows how the
*Armillaria* isolates from the Penja pepper fields grouped to-
gether into a single phylogenetic clade alongside the type
*A. camerunensis* and isolates from Cameroon associated with
cacao root rot. This clade is sister to another clade representing
*A. fuscipes* (Fig. 2). This result is similar to those of Coetzee
et al. (2005) who, relying on ITS and IGS1 based phylogenies,
split *A. fuscipes sensu lato* (s. l.), at the time considered a
single specific entity, into two distinctive sister species. One
species represented *A. fuscipes sensu stricto* (s. s.), and includ-
ed isolates from South Africa, Zimbabwe, Kenya, and La
Reunion. The second species, to which the Penja isolates
appear to belong to, represented an undescribed species and
included isolates from Cameroon, Zambia, and Zimbabwe.
When Roux et al. (2012) used IGS1 phylogeny to identify
*Armillaria* isolates associated with cacao root rot in
Cameroon; they concluded that these isolates were conspecif-
ic with the unnamed second clade of *A. fuscipes* s. l. This clade
was later described as *A. camerunensis* by Koch et al. (2017).
Unlike with the ITS as seen in this study, the phylogenetic tree
based on IGS1 shows substantial sub-structuring in the
*A. camerunensis* clade in Cameroon (Roux et al. 2012), which
could be indicative of a high genetic diversity and possibly
cryptic speciation in this species. Although two other

*Armillaria* species have in the past been reported from
Cameroon, including *A. elegans* and *A. heimii* (*A. fuscipes*)
(Hennings 1895; Roux et al. 2012; Koch et al. 2017), DNA
sequence studies have to date only recovered *A. camerunensis*
in the country.

Many of the farms had previously grown, or were still
growing, crops like cacao, *Coffea* spp. or fruit trees such as
*Citrus* species, avocado (*Persea americana*), African plum
(*Dacryodes edulis*) or cola nut (*Cola nitida*), next to or asso-
ciated with the pepper (Table 2). Certain plots contained forest
trees or remnants (stumps) thereof. Many of these are potential
hosts for *Armillaria* (Roux and Coetzee 2016), which would
explain the presence of the pathogen. *Armillaria* has been
reported in Cameroon as early as 1892 (Hennings 1895) and
has been found on cocoa in Cameroon, apparently as early as
1907 (Roux and Coetzee 2016), as well as in several other
West African countries such as Sao Tomé, Togo and Ghana
(Dade 1927; Rishbeth 1980; Roux and Coetzee 2016).

Since *Armillaria* spp. may survive decades in stumps and
roots in the soils (Roux and Coetzee 2016), preparation of the
terrain where a pepper plantation is to be established is key.
Generally speaking, this disease spreads *via* root to root con-
tact between infected and healthy plants, or through
rhizomorphs or by mycelium present in the soil (Denman
et al. 2000). Thus, manual removal of potentially infected
plant material such as stumps and their root systems before
establishment of the pepper plantation is recommended. Soil
fumigation and soil inoculation with antagonistic fungi (e.g.
Otieno et al. 2003) could further help to prepare a terrain.
However, the efficacy of these methods is certainly not a hun-
dred percent.

Once the disease is present, trenching is often used to pre-
vent spread (Worrall 2004). This is only successful if the
trench is dug well beyond the last affected trees. In one
farmer's field in the Njombe-Penja district they tried this ap-
proach, which unfortunately did not seem to be very success-
ful. This was either because the trench was dug too close to the
area that was visibly affected or, since the affected area was on
a slope, there was a downward movement of infectious prop-
agules that was not stopped by the trench.

Many Penja pepper farmers use fungicides to control the
disease cause by *A. camerunensis*, as this is the principal
means they are comfortable with and knowledgeable about
when it comes to pest and disease control. Unfortunately,
chemical control is not very efficient in controlling
Armillaria root rot (Thomidis and Exadaktylou 2012; Roux
and Coetzee 2016).

Since the disease was more prevalent and more clearly
visible on *S. mombin* than on pepper vines, we hypothesize
that generally speaking the disease spreads through direct root
contact from *S. mombin* to *P. nigrum*. If so, potentially using a
different support tree not affected by *Armillaria* could solve
the problem. Otherwise, support systems such as metal or
<figure><img src="image_5.png" /><figcaption>**Fig. 2** Maximum Likelihood (ML) tree inferred from the ITS region showing the phylogenetic position of isolates obtained from *Piper nigrum* and *Spondias mombin* (in bold) from Cameroon. The tree was</figcaption></figure>

rooted with sequences of *Armillaria hinnulea* (KX618553 and KX618555). Bootstrap values ≥70% from 1000 replicates are displayed at the nodes
concrete poles could be considered, although this would require an investment out of reach of most farmers. However, since a few pepper vines with clear *Armillaria* disease symptoms were observed attached to seemingly healthy support trees, this raises the question whether the disease can spread from *P. nigrum* to *S. mombin*. Ultimately, it seems that both species are potential hosts of this *Armillaria* sp.

Based on the results presented here, it is clear that Armillaria root rot, caused by *A. camerunensis*, is one of the main phytosanitary problems of Penja pepper cultivation, causing considerable economic impact. Familiarization of farmers with the causal agent and appropriate control methods are urgently needed to ensure sustained production of highly valued Penja Pepper. This is the first report of Armillaria root rot of *S. mombin* and *P. nigrum* globally.

**Acknowledgements** The authors would like to thank the Penja Pepper Geographical Indication Group (IGPP) producers, in particular René Claude Metomo Elogo (President of Association) and Emmanuel Nzenowo (Executive Secretary) for allowing database and field access. We also thank Fokou Rodrigue, Diazem Clovis, Tocké, Ngonga Florian, and Njonkam Ferdinand, agricultural technicians who assisted in the surveys. Funding and resources were provided by CIRAD, IRAD, the University of Pretoria and through the “Debt reduction-development contract (C2D) France Cameroon” Poivre de Penja project. The authors were free to publish any or all results originating from their work.

**Author contributions** Conceptualization: Petchayo TS, M Ndoumbé Nkeng, GM ten Hoopen; Methodology: Petchayo TS, JM Tchotet Tchoumi, M Ndoumbé Nkeng, GM ten Hoopen; Investigation: Petchayo TS, JM Tchotet Tchoumi, M Mbenoun, GM ten Hoopen; Formal analysis: Petchayo TS, JM Tchotet Tchoumi, J Roux, M Mbenoun, M Ndoumbé Nkeng, GM ten Hoopen; Resources: J Roux, GM ten Hoopen; Funding acquisition: CV Mfegue, GM ten Hoopen; Project administration: S Nyassé; Supervision: J Nguefack, BAD Begoude, GM ten Hoopen; Writing original draft: Petchayo TS, GM ten Hoopen; Manuscript revision: Petchayo TS, GM ten Hoopen, M Mbenoun, JM Tchotet Tchoumi, J Roux. All authors read and approved the final manuscript.

## Compliance with ethical standards

**Conflict of interest** On behalf of all authors, the corresponding author states that there is no conflict of interest.

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