bioRxiv preprint doi: https://doi.org/10.1101/2020.10.07.329532; this version posted October 7, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 1

1 Characterisation of Metarhizium majus (Hypocreales:

2 Clavicipitaceae) isolated from the Western Cape province,

3 South Africa

4

5 Letodi L. Mathulwe1, Karin Jacobs2, Antoinette P. Malan1*, Klaus Birkhofer3, Matthew F.

6 Addison1, Pia Addison1

7

8 1 Department of Conservation Ecology and Entomology, Faculty of AgriSciences, Private

9 Bag X1, Matieland 7602, Stellenbosch, South Africa

10 2 Department of Microbiology, Faculty of Science, Private Bag X1, Matieland 7602,

11 Stellenbosch, 7602, South Africa

12 3 Department of Ecology, Brandenburg University of Technology, Cottbus, Germany

13

14

15 *Corresponding author

16 E-mail: [email protected] (APM)

17 bioRxiv preprint doi: https://doi.org/10.1101/2020.10.07.329532; this version posted October 7, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 2

18 Abstract

19 Entomopathogenic fungi (EPF) are important soil-dwelling entomopathogens, which can be

20 used as biocontrol agents against pest insects. During a survey of the orchard soil at an organic

21 farm, the EPF were identified to species level, using both morphological and molecular

22 techniques. The EPF were trapped from soil samples, taken from an apricot orchard, which

23 were baited in the laboratory, using susceptible host insects. The identification of Metarhizium

24 majus from South African soil, using both morphological and molecular techniques, is verified.

25 The occurrence of M. majus in the South African soil environment had not previously been

26 reported.

27

28 Keywords: Entomopathogenic fungi; Metarhizium majus; Morphological; Molecular;

29 Biological control bioRxiv preprint doi: https://doi.org/10.1101/2020.10.07.329532; this version posted October 7, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 3

31 Introduction

32 Entomopathogenic fungi (EPF), which are cosmopolitan components of the soil microbiota,

33 are commonly isolated from the soil environment, for use as biological control agents to

34 manage a broad range of pest insects [1,2]. The genus Metarhizium Sorokin (Ascomycetes,

35 Hypocreales) consists of asexually reproducing EPF species, which are characterised by the

36 production of green conidia on the surfaces of infected insect cadavers, and when they are

37 grown on a growth medium [3]. Species belonging to the genus Metarhizium are well-studied

38 entomopathogens, which are widely commercialised. Many products derived from the species

39 are on the market, for use against a wide range of economically-important insect pests [4,5].

40 Distinguishing between different Metarhizium species morphologically is based on their

41 conidial morphology, as using other morphological characteristics is challenging [3].

42 Metarhizium species are mainly identified, and differentiated from each other, using molecular

43 techniques [6]. Two main monophyletic groups fall within the Metarhizium anisopliae species

44 complex. The PARB clade consists of Metarhizium pinghaense Chen & Guo, Metarhizium

45 anisopliae sensu stricto, Metarhizium robertsii (Metchnikoff) Sorokin and Metarhizium

46 brunneum Petch, while the MGT clade consists of Metarhizium majus Johnst., Bisch., Rehner

47 and Humber and Metarhizium guizhouense Chen and Guo [3,7]. The MGT species are

48 distinguished from the PARB clade by their relatively large conidia, with M. majus having the

49 larger cylindrical conidia relative to M. guizhouense, which possess the second largest conidia

50 [7]. Metarhizium majus and M. guizhouense have been differentiated from each other, based

51 on molecular data, using the translation elongation factor-1 alpha gene (EF-1α) [3,7].

52 In the current study, additional regarding the morphological and molecular evidence is

53 provided to present the first report on the occurrence of Metarhizium majus in South African

54 soil. bioRxiv preprint doi: https://doi.org/10.1101/2020.10.07.329532; this version posted October 7, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 4

55 Materials and Methods

56 Collection of soil samples and EPF baiting

57 Soil samples were collected from the orchards surveyed, at a depth of 15 cm, from under the

58 tree canopy on Tierhoek farm (-33˚13'19.687''S 19˚38'44.281'') (-33,711596; 19,790730), near

59 Robertson in the Western Cape province. After an initial sifting, each soil sample was

60 transferred to 1-L plastic containers, baited with the last-instar larvae of the wax moth Galleria

61 mellonella L. (Lepidoptera: Pyralidae) and Tenebrio molitor (Coleoptera: Tenebrionidae), kept

62 for 14 days at a room temperature of 25ºC [8,9,10]. The soil samples were everted after every

63 3 days, so as to ensure the penetration of the insect bait into the soil. After every 7 days, the

64 dead insects that showed EPF infection, which was observed in the form of hardening, or overt

65 mycosis of the insect cadaver, were removed from the soil samples. To check the cause of

66 mortality, the dead insects, after first being washed in sterile distilled water, were then dipped

67 in 75% ethanol, followed by them being dipped twice in distilled water. Each dead insect was

68 placed in a Petri dish fitted with moist filter paper. The Petri dishes were then placed in 2-L

69 plastic containers, fitted with paper towels moistened using sterile distilled water, and

70 incubated at room temperature.

71 Following a further 7 days of incubation, the spores from the surface of the dead insect

72 cuticles were placed on a Sabouraud dextrose agar plate with 1 g of yeast extract (SDAY),

73 supplemented with 200 µl of Penicillin-Streptomycin, so as to prevent bacterial contamination.

74 After the SDAY plates were sealed and incubated at 25ºC, they were checked for fungal growth

75 for a period of two weeks. The pathogenicity against insects was verified, using the larvae of

76 the wax moth [11].

77 Morphological identification bioRxiv preprint doi: https://doi.org/10.1101/2020.10.07.329532; this version posted October 7, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 5

78 Temporary slides were prepared by means of trapping spores in a drop of water on a glass slide

79 with a cover slip, secured with glyceel. The size of the conidia was determined, measuring both

80 the length and the width of 30 spores, using a Zeiss Axiolab 5 light microscope, equipped with

81 an Axiocam 208 camera. SEM preparation of spores of different Metarhizium species

82 including M. majus, M. robertsii (GenBank accession number MT378171), M. pinghaense

83 (MT895630) and M. brunneum (MT380848) were prepared and photographed by the Central

84 Analytical Facility of the Stellenbosch University.

85 Molecular identification

86 For molecular identification, the fungal DNA was extracted from culture plates, using a Zymo

87 research Quick-DNA fungal/bacterial miniprep kit, according to the manufacturer’s protocol.

88 The polymerase chain reaction was conducted, using the KAPA2G ReadyMix PCR kit.

89 Characterisation was based on sequencing of the ITS region and two genes, consisting of the

90 internal transcribed spacer (ITS) (primers ITS1and ITS4), the partial tubulin (BtuB) (primers

91 Bt2a and Bt2b) and the partial EF-1α (primers EF1F and EF2R) [12,13]. The PCR thermocycle

92 conditions accorded with the technique used by [14]. The PCR products were visualised on an

93 agarose gel, using ethidium bromide. The sequences, which were generated by the Central

94 Analytical Facility at Stellenbosch University, were aligned and edited using the CLC main

95 workbench (ver. 8) and blasted on the GenBank database of the National Centre for

96 Biodiversity Information (NCBI) for identification. Fungal cultures were deposited at the

97 Agricultural Research Council (ARC), the Biosystematics Division, Pretoria fungal collection.

98 Phylogenetic analyses

99 Phylogenetic analyses were conducted using the dataset from Rehner and Kepler (2017) [13]

100 and Luz et a. (2019) [12] , combining sequences of three loci (ITS, BT, tef1), so as to identify

101 the species concerned. The alignments were done employing ClustalX, using the L-INS-I bioRxiv preprint doi: https://doi.org/10.1101/2020.10.07.329532; this version posted October 7, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 6

102 option. The software package PAUP [15] was used to construct a neighbour-joining

103 phylogenetic tree, using a bootstrap analysis of 1 000 replicates. A Bayesian analysis was run

104 using MrBayes ver. 3.2.6 [16]. The analysis included four parallel runs of 200 000 generations,

105 with a sampling frequency of 200 generations. The posterior probability values were calculated

106 after the initial 25% of the trees were discarded. The fungal isolates used in the current study

107 to construct the phylogenetic trees are listed in Table 1.

108 bioRxiv preprint doi: https://doi.org/10.1101/2020.10.07.329532; this version posted October 7, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 7

109 Table 1. Reference of Metarhizium species isolates used in phylogenetic analyses and their

110 culture number, isolation source, country of origin and GenBank accession numbers.

Species Culture number Origin Country 5'TEF BTUB ITS M. acridum ARSEF 324 Orthoptera Australia EU248844 EU248812 HM055449 M. acridum ARSEF 7486b Orthoptera Niger EU248845 EU248813 NR_132019 M. alvesii CG1123b Soil Brazil KC520541 - - M. anisopliae ARSEF 6347 Homoptera Colombia EU248881 - - M. anisopliae ARSEF 7450 Coleoptera Australia EU248852 EU248823 HQ331464 M. anisopliae ARSEF 7487b Orthoptera Ethiopia DQ463996 EU248822 HQ331446 M. anisopliae CHE CNRCB 235 Hemiptera Mexico KU725694 - - M. anisopliae ESALQ 1614 Soil Brazil KP027962 - - M. anisopliae ESALQ 1617 Soil Brazil KP027957 - - M. brunneum ARSEF 2107b Coleoptera USA EU248855 - - M. brunneum ARSEF 4179 Soil Australia EU248854 EU248825 HQ331451 M. frigidum ARSEF 4124b Coleoptera Australia DQ463978 EU248828 NR_132012 M. guizhouense ARSEF 6238 Lepidoptera China EU248857 EU248830 HQ331447 M. guizhouense CBS 258.90b Lepidoptera China EU248862 EU248834 HQ331448 M. humberi IP 1 Soil Brazil, GO JQ061188 - - M. humberi IP 16 Soil Brazil, GO JQ061196 - - M. humberi IP 41 Soil Brazil, GO JQ061199 - - M. humberi IP 46b Soil Brazil, GO JQ061205 - - M. kalasinense BCC53581 Coleoptera Thailand KX823944 - - M. kalasinense BCC53582b Coleoptera Thailand KX823945 - - M. lepidiotae ARSEF 7412 Coleoptera Australia EU248864 EU248836 HQ331455 M. lepidiotae ARSEF 7488b Coleoptera Australia EU248865 EU248837 HQ331456 M. majus ARSEF 1914b Coleoptera Philippines KJ398801 KJ398571 HQ331445 M. majus ARSEF 1946 Coleoptera Philippines EU248867 EU248839 - M. pingshaense CBS 257.90b Coleoptera China EU248850 EU248820 HQ331450 M. pingshaense ARSEF 4342 Coleoptera Solomon Islands EU248851 EU248821 HQ331454 M. robertsii ARSEF 23 Coleoptera USA KX342726 - - M. robertsii ARSEF 727 Orthoptera Brazil DQ463994 - - M. robertsii ARSEF 4739 Soil Australia EU248848 - - M. robertsii ARSEF 7501 Coleoptera Australia EU248849 - - M. robertsii ESALQ 1621 Soil Brazil KP027980 - - M. robertsii ESALQ 1625 Soil Brazil KP027974 - - M. robertsii ESALQ 1634 Soil Brazil KP027971 - - M. robertsii ESALQ 1635 Soil Brazil KP027977 - - M. majus TH152 Soil South Africa MT330376 MT330375 MT254988 111

112 bioRxiv preprint doi: https://doi.org/10.1101/2020.10.07.329532; this version posted October 7, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 8

113 Results

114 Morphological identification

115 The growth pattern of M. majus on SDAY medium was found to typify the genus Metarhizium

116 (Fig 1A). The conidia of the mature colonies, which were dark green, formed chains of equal

117 length in clusters (Fig B and C). The conidia, which were oval in shape (n = 30), varied 9.0

118 (7.5–10.2) µm in length and 4.3 (4.0–4.5) µm in width (Fig 1 D). The conidiophores of M.

119 majus were branched, with the apices of the branches bearing from one to several elongated

120 phialides (Fig E and F). SEM pictures of the four different Metarhizium species showed no

121 morphological difference in the surface pattern from those of M. majus (Fig 1 G and H).

122 Fig 1. Morphology of Metarhizium majus TH152, (A) a three-week-old culture on SDAY

123 medium; (B) spores on older plates; (C) bundles of spore strings of the same length; (D)

124 spore shape and size; (E, F) mature phialides with conidiogenous cells and conidia; (G,

125 H) scanning electron microscope picture showing the surface of the conidia.

126 Molecular identification

127 Sequences generated for the Metarhizium majus strain collected from an apricot orchard

128 corresponded to those of the type strains. Using the BLAST function, the ITS region cannot

129 differentiated M. majus from M. anisopliae, however the elongation factor and the beta-tubulin

130 gene sequences confirm the species to be M. majus. Sequences were deposited in the GenBank

131 (ITS: MT254988, EF: MT330376, beta-tubulin: MT330375).

132 Phylogenetic analysis

133 The neighbour-joining phylogeny of the concatenated dataset resulted in a high degree of

134 support for the monophyly of the MGT clades (Fig 2). The M. guizhouense was found to form bioRxiv preprint doi: https://doi.org/10.1101/2020.10.07.329532; this version posted October 7, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 9

135 a sister group with M. majus, with high percentages of bootstrap support of 87% (Fig 2) and

136 82% (Fig 3), respectively. The MGT clade forms a sister clade to the PARB clade (Fig 2). The

137 local M. majus TH152 isolate, and the two M. majus isolates (ARSEF 1914b and ARSEF 1946)

138 collected in the Philippines (Fig 3), grouped in the same clade, with 100% bootstrap

139 confidence.

140 Fig 2. Neighbour joining likelihood phylogenetic tree of Metarhizium majus related to the

141 PARB and MGT clades from the analysis of concatenate datasets of 5'intron-rich region

142 of the ITS and the translation elongation factor-1alpha (EF-1α). Bootstrap values are

143 denoted above branches. The tree was rooted, using the sequence from Metarhizium

144 brasiliense ARSEF2948b as outgroup and isolates with T = indicate the type strain.

145 Fig 3. Neighbour-joining phylogenetic tree of Metarhizium majus with regards to related

146 species, based on analysis of the 5'intron-rich region of the translation elongation factor

147 1-alpha (EF-1α) gene sequences. The tree was rooted using the sequence from

148 Metarhizium frigidum ARSEF 4124b as the outgroup. Bootstrap values are denoted above

149 branches and isolates with T = indicate the type strain.

150 Discussion

151 The genus Metarhizium consists of a diverse group of entomopathogenic species, having a

152 cosmopolitan distribution and a wide range of insect hosts. Metarhizium majus is considered

153 to be an important potential biological control agent for various insect pests. The fungus is

154 deemed to be an effective biological agent, when it is used against Odoiporus longicollis

155 Olivier (Coleoptera: Curculionidae), the banana pseudostem weevil, which is a serious pest

156 affecting banana production [18,2]. The EPF is also used to manage Oryctes rhinoceros L.

157 (Coleoptera: Scarabaeidae), the coconut rhinoceros beetle, the activities of which result in

158 major crop losses in coconut and palm oil plantations [19,20]. bioRxiv preprint doi: https://doi.org/10.1101/2020.10.07.329532; this version posted October 7, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 10

159 An isolate of M. majus was obtained from soil samples, during a survey of EPF in an

160 apricot orchard. The morphological evidence obtained supported the isolate as being M. majus,

161 especially in terms of the size of the conidia, which are the largest of all of the Metarhizium

162 species. The growth of the hypha is the same for the genus, therefore, being difficult to

163 distinguish from the other species. A previous study indicated that M. majus forms one of the

164 species in the group with the largest conidia, ranging from 8.5 to 14.5 µl in length and from 2.5

165 to 3.0 µl in width, with such a characteristic usually being the only usable morphological

166 difference in the group [3,21]. The surface structure of M. majus was found not to be visually

167 different from four different species of Metarhizium.

168 The presence of M. majus in the soil environment has previously been recorded in other

169 countries, like Japan [7] the USA [2], Australia [22] and Denmark [23]. However, it is the first

170 time that the EPF species concerned has been isolated from South African soil, with the current

171 study providing both morphological and molecular evidence of the presence of M. majus in

172 such soils. The discovery of the South African M. majus isolate adds new information to the

173 body of knowledge regarding soil fungal biodiversity. The presence of M. majus in South

174 Africa also increases the number of available local EPF isolates that can be used in agricultural

175 ecosystems for the management of insect pests, as the isolates possess entomopathogenic

176 properties. Its potential as a biocontrol agent, of especially Coleoptera, with be investigated in

177 future studies.

178 Acknowledgments

179 The authors are very grateful to the owner of Tierhoek organic farm. We also wish to thank the

180 all staff and students of the Stellenbosch University participating in the soil sampling.

181 Conflict of interest bioRxiv preprint doi: https://doi.org/10.1101/2020.10.07.329532; this version posted October 7, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 11

182 All authors declare that there is no conflict of interest in the article.

183 Author Contributions

184 Conceptualization: Klaus Birkhofer, Pia Addison, Matthew Addison.

185 Formal analysis: Letodi Mathulwe, Karin Jacobs.

186 Funding acquisition: Pia Addison, Klaus Birkhofer.

187 Investigations: Letodi Mathulwe, Karin Jacobs, Antoinette Malan.

188 Project administration: Pia Addison, Klaus Birkhofer.

189 Supervision: Pia Addison.

190 Writing - original draft: Letodi Mathulwe, Karin Jacobs, Antoinette Malan.

191 Writing - review & editing: Letodi Mathulwe, Karin Jacobs, Antoinette Malan, Klaus

192 Birkhofer, Pia Addison, Matthew Addison

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270 https://doi.org/10.1080/21501203.2018.1524799 bioRxiv preprint doi: https://doi.org/10.1101/2020.10.07.329532; this version posted October 7, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. bioRxiv preprint doi: https://doi.org/10.1101/2020.10.07.329532; this version posted October 7, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. bioRxiv preprint doi: https://doi.org/10.1101/2020.10.07.329532; this version posted October 7, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.