Universidade Federal de Santa Maria

Ci. Fl., Santa Maria, v. 31, n. 2, Apr./June, 2021

DOI: 10.5902/1980509837118

ISSN 1980-5098

Submitted: 28/02/2019 • Approved: 20/10/2020 • Published: 1st/06/2021

Artigos

Host status of plants from the Cerrado Biome to Meloidogyne spp.

Hospedabilidade de plantas do Bioma Cerrado a Meloidogyne spp.

Reinaldo Rodrigues PimentelI

Ramon Lira dos AnjosII

Adriana Andrade FerreiraIII

Gisele Pereira DomicianoIV

Regina Célia de OliveiraV

Cleber FurlanettoVI

I Agronomist, Dr., Universidade de Brasília, Brasília, DF, Brazil

https://orcid.org/0000-0003-4319-950X – reinaldorodriguesagro@hotmail.com

II Agronomist, Dr., Universidade de Brasília, Brasília, DF, Brazil

https://orcid.org/0000-0002-7488-7842 – ramon.unb@gmail.com

III Biomedic, MSc., Embrapa Recursos Genéticos e Biotecnologia, Brasília, DF, Brazil

https://orcid.org/0000-0002-9486-789X – adrianabiomedica1@gmail.com

IV Agronomist, Dr., Universidade de Brasília, Brasília, DF, Brazil

https://orcid.org/0000-0003-4389-3585 – gpdomiciano@yahoo.com.br

V Biologist, Dr., Universidade de Brasília, Brasília, DF, Brazil

https://orcid.org/0000-0001-7891-2630 – reginacelia@unb.br

VI Agronomist, Dr., Universidade de Brasília, Brasília, DF, Brazil

https://orcid.org/0000-0003-0575-0487 – cfurla@hotmail.com

ABSTRACT

Among the Brazilian biomes, Cerrado is the second in territorial extension and diversity of plants and animal species. The Cerrado’s biodiversity has been studied in terms of its flora and fauna, lacking data from microorganisms representing the microfauna, such as root-knot nematodes. Meloidogyne spp. are sedentary endoparasitic nematodes of plants. Its presence in soil covered with native cerrado vegetation, under preservation, is a strong indication that its permanence occurs due to the parasitism of native plants. This work aimed to study the host suitability of cerrado plants to Meloidogyne spp. Thirty-five plant species from the Cerrado biome, including grasses and trees, were selected for inoculation with four Meloidogyne isolates, three of them previously collected in a native Cerrado area under preservation (Meloidogyne incognita, Meloidogyne javanica, and Meloidogyne morocciensis), and the fourth (Meloidogyne paranaensis) an exotic species in Cerrado. Five thousand eggs and second-stage juveniles (J2) of each nematode species were inoculated per plant. The experiments were conducted with 35 treatments (native plants) and 5 replicates, having been repeated in time. The variables assessed were gall index and egg mass index, eggs and J2 per gram of root and reproductive factor (RF), as well as the symptomatology caused by the nematodes on the roots. The following plant species were considered as hosts by presenting mean of the Reproduction Factor (RF)>1.0: Triplaris gardneriana for Meloidogyne javanica, Meloidogyne incognita, and Meloidogyne morocciensis; Andropogon bicornis for Meloidogyne javanica and Meloidogyne incognita and Copaifera langsdorffii for Meloidogyne incognita; The following plant species were classified as potential hosts by developing RF>1.0 in at least one of the replicates: Copaifera langsdorffii for Meloidogyne morocciensis; Esenbeckia leiocarpa for Meloidogyne javanica and Guibourtia hymenifolia for Meloidogyne incognita. The other plant species were classified as non-hosts considering the conditions of this study. The inoculated plants showed varied symptoms on the roots, such as galls associated with egg masses, egg masses without galls, swelling with egg masses, cracks with egg masses without galls, and swelling with egg masses.

Keywords: Root-knot nematodes; Brazilian savanna; Native plants; Host plants

RESUMO

Dentre os biomas brasileiros, o Cerrado é o segundo em extensão territorial e em diversidade de espécies vegetais e animais. A biodiversidade do Cerrado tem sido estudada no que tange a sua flora e fauna, carecendo de dados de microorganismos representantes da microfauna, a exemplo dos nematoides de galhas radiculares. Meloidogyne spp. são nematoides endoparasitas sedentários de plantas. A sua presença em solo de áreas com vegetação nativa de cerrado, sob preservação, é um forte indício de que a sua permanência ocorre devido ao parasitismo de plantas nativas. Este trabalho objetivou estudar a hospedabilidade de plantas ocorrentes no bioma cerrado a Meloidogyne spp. Foram selecionadas trinta e cinco espécies vegetais desse bioma, incluindo espécies gramíneas e arbóreas, para estudo da hospedabilidade frente a três isolados de Meloidogyne coletados em área nativa de cerrado sob preservação (Meloidogyne incognita, Meloidogyne javanica e Meloidogyne morocciensis), além de uma espécie até então exótica ao cerrado (Meloidogyne paranaensis). Foram inoculados 5.000 ovos e eventuais juvenis de segundo estádio (J2) de cada espécie de nematoide por planta. Os experimentos foram conduzidos com 35 tratamentos (plantas nativas) e 5 repetições, tendo sido repetidos no tempo. As variáveis avaliadas foram índice de galhas e de massas de ovos, ovos e J2 por grama de raiz e fator de reprodução, além da sintomatologia causada pelos nematoides nas raízes. As seguintes espécies vegetais foram consideradas hospedeiras, com média do Fator de Reprodução (FR)>1,0: Triplaris gardneriana para Meloidogyne javanica, Meloidogyne incognita e Meloidogyne morocciensis; Andropogon bicornis para Meloidogyne javanica e Meloidogyne incognita e Copaifera langsdorffii para Meloidogyne incognita. As seguintes espécies vegetais foram classificadas como hospedeiras potenciais, com FR>1,0 para pelo menos uma das repetições: Copaifera langsdorffii para Meloidogyne morocciensis; Esenbeckia leiocarpa para Meloidogyne javanica e Guibourtia hymenifolia para Meloidogyne incognita. As demais espécies vegetais foram classificadas como não hospedeiras para as condições deste estudo. As plantas inoculadas apresentaram sintomas variados nas raízes como a presença de galhas associadas a massas de ovos, formação de massas de ovos sem galhas, rachaduras com massas de ovos e ausência de galhas e intumescimento com massas de ovos.

Palavras-chave: Nematoide das galhas; Savana brasileira; Plantas nativas; Plantas hospedeiras

1 INTRODUCTION

The Brazilian Cerrado boasts one of the richest biodiversities. It also presents agriculture in rapid expansion, contributing significantly to the growth of the Brazilian agribusiness and strength of the country’s trade balance. This scenario is often challenged by the incidence of phytopathogens, especially phytoparasitic nematodes that reduce crop productivity. Economical losses caused by nematodes on the global agriculture are estimated in 157 billion dollars/year (ABAD et al., 2008; ONKENDI et al., 2014).

Among phytoparasitic nematodes, species of the genus Meloidogyne are the major cause of economic damage, especially Meloidogyne incognita, Meloidogyne javanica and Meloidogyne arenaria in a tropical climate and Meloidogyne hapla in a temperate climate (ONKENDI et al., 2014). However, emerging species such as Meloidogyne paranaensis and Meloidogyne enterolobii have gained importance over the years due to their spread and damage to crops of increasing economic importance such as coffee and guava.

Gall-inducing nematodes of the genus Meloidogyne have been reported to occur in soils of cerrado areas under permanent preservation (SOUZA; DOLINSKI; HUANG, 1994; SILVA et al., 2013). According to Antes et al. (2012) and Silva et al. (2013), Meloidogyne species occur in low population density in soils occupied with native vegetation, surviving due to the interaction with plant species. According to the same authors, the low population density of nematodes in areas occupied with native vegetation can be explained by the diversity and the genetic variability of vegetation present in those areas. However, their infective and reproductive capacity on plants of natural biomes is still poorly understood. This work aimed to study the reproduction of isolates of Meloidogyne incognita, Meloidogyne javanica, Meloidogyne morocciensis and Meloidogyne paranaensis in plant species of the Cerrado biome.

2 MATERIAL AND METHOD

2.1 Origin, identification, and maintenance of Meloidogyne spp.

The populations of Meloidogyne spp. used in this study were collected in Cerrado areas under permanent preservation in Brasília, Federal District, according to a survey conducted by Silva et al. (2013). Meloidogyne paranaensis, was obtained from the Embrapa-Cenargen collection. Meloidogyne morocciensis was collected in clean field phytophysiognomy of Brasília National Park, Meloidogyne incognita in gallery forest, and Meloidogyne javanica in the clean field at ‘Água Limpa’ farm of the University of Brasília. The Meloidogyne species mentioned above were confirmed based on the esterase phenotypes, according to the methodology proposed by Alonso and Alfenas (1998) as follows: Meloidogyne javanica (EST-J3), Meloidogyne incognita (EST-I1), Meloidogyne morocciensis (EST-A3), and Meloidogyne paranaensis (EST-P1). The four nematode species were inoculated in tomato cv. Santa Clara and maintained in a greenhouse.

2.2 Plant seeds and seedlings

Seeds from tree plants, whose origin is unknown, were provided by the Nursery II of NOVACAP Company (‘Companhia Urbanizadora da Nova Capital do Brasil’). For Enterolobium gummiferum (Tamboril), the seeds were collected from a single tree in a Cerrado area. Grasses were multiplied in pots by cultivating tillers collected in the field. Exsiccates were made for each botanical species and deposited at the Herbarium of the University of Brasília to confirm the species identity.The tree plants were transplanted from tubes to polyethylene bags with a capacity for 2500 cm3 of a sterile substrate and the grasses were transplanted to plastic pots with a capacity for 3 L of the substrate, which was composed of soil and sand in a ratio of 1:1. The plants were kept in a greenhouse at the Biology Experimental Station of the University of Brasília until they reached 30 cm in height. A total of thirty-five plant species from the Cerrado biome were studied (Table 1).

Table 1 – Botanical species from the Cerrado biome evaluated for hostability to Meloidogyne spp.

Tabela 1 – Espécies botânicas ocorrentes no bioma Cerrado avaliadas quanto à hospedabilidade a Meloidogyne spp.

Scientific name

Common name

Type

Family

Albizia niopoides (Benth.) Burkart

Angico-branco

Tree

Fabaceae

Anacardium humili St. Hilaire

Cajuí

Tree

Anacardiaceae

Anadenanthera macrocarpa (Benth.) Brenan

Angico-preto

Tree

Fabaceae

Astronium flaxinifolium Schott

Gonçalo-Alves

Tree

Anacardiaceae

Andropogon bicornis L.

Andropogon

Grass

Poaceae

Calophyllum brasiliense Cambess.

Landim

Tree

Calophyllaceae

Copaifera langsdorffii Desf.

Copaíba

Tree

Fabaceae

Cybistax antisyphilitica Mart.) Mart.

Ipê-verde

Tree

Bignoniaceae

Dalbergia miscolobium Benth.

Caviúna

Tree

Fabaceae

Jacaranda mimosifolia D. Don

Jacarandá-Mimoso

Tree

Fabaceae

Echinolaena inflexa (Poir.) Chase.

Capim-flechinha

Grass

Poaceae

Elionurus muticus (Spreng.) Kuntze.

Capim-cheiroso

Grass

Poaceae

Enterolobium gummiferum (Mart.) J.F. Macbr.

Tamboril

Tree

Fabaceae

Eriochrysis filiformis (Hack.) Filg.

-

Grass

Poaceae

Eriotheca pubescens Mart. & Zucc.

Paineira-loira

Tree

Malvaceae

Esenbeckia leiocarpa Engl.

Guarantã

Tree

Rutaceae

Eugenia tomentosa Camb.

Cabeludinha

Tree

Myrtaceae

Guibourtia hymenifolia (Moric.) J. Leonard

Copaibeira

Tree

Fabaceae

Handroanthus impetiginosus (Mart. ex DC.) Mattos

Ipê-roxo

Tree

Bignoniaceae

Handroanthus caraíba (Mart.) Mattos.

Ipê-caraíba

Tree

Bignoniaceae

Handroanthus chrysotrichus (Mart. ex DC.)

Ipê-tabaco

Tree

Bignoniaceae

Handroanthus serratifolius (Vahl) S. O. Grose

Ipê-amarelo

Tree

Bignoniaceae

Hymenaea courbarilvar. stilbocarpa L.

Jatobá

Tree

Fabaceae

Inga fagifolia Benth.

Ingá-mirim

Tree

Fabaceae

Inga heterophylla Willd.

Ingá-colar

Tree

Fabaceae

Kielmeyera coriaceae Mart. & Zucc.

Pau-santo

Tree

Calophyllaceae

Kielmeyera speciosa A. St.-Hil.

Pau-santo

Tree

Calophyllaceae

Myracrodruon urundeuva Fr. All.

Aroeira preta

Tree

Anacardiaceae

Paspalum pilosum Lam.

Paspalum

Grass

Poaceae

Paspalum plicatulum Michx.

Plicatulum

Grass

Poaceae

Physocalymma scaberrimum Pohl

Pau-de-rosas

Tree

Litraceae

Pseudobombax tomentosum (Mart.) A. Robyns

Imbiruçu

Tree

Malvaceae

Stryphnodendron adstringens (Mart.) Coville

Barbatimão

Tree

Fabaceae

Terminalia tomentosum L.

Piúna

Tree

Combretaceae

Triplaris gardneriana Wedd.

Pajeú

Tree

Polygonaceae

Source: Authors (2019)

2.3 Physical-chemical and biological analysis of the substrate

The chemical analysis of the substrates used in the greenhouse tests was carried out by the company ‘Solocria Laboratório Agropecuário LTDA’, located in Goiânia – Goiás state. Soil samples were collected from the rizosphere of grasses during tiller collection and analyzed for the presence of phytoparasitic nematodes, according to the methodology proposed by Jenkins (1964).

2.4 Reaction of Cerrado plants to Meloidogyne spp.

2.4.1 Experimental design, inoculum and evaluated variables

Two tests were carried out at different times for each plant species, being them established in a completely randomized design (CRD), with five replications. Single plants were inoculated with five thousand eggs and eventual J2 of each Meloidogyne species. The inoculated plants were kept on separate benches to avoid contamination. The tomato cv. ‘Santa Clara’ was used as a control treatment due to the high susceptibility to Meloidogyne spp. The plant species used in the tests were free from nematode contamination.

2.4.2 Root staining and symptomatology

Plant shoots were separated from the roots, washed in tap water and immersed in acid fuchsin solution 0.1% for seven minutes to color the egg masses, according to the methodology proposed by Silva, Santos and Ferraz (1988). Egg masses were counted using a light microscope (DICKSON; STRUBLE, 1965). Symptoms produced by the nematodes on plant roots were described and photo-documented.

2.4.3 Egg Mass Index, Gall Index, Number of Eggs and / or J2 per Gram of Root and Reproduction Factor

To determine the EMI (Egg Mass Index), the Taylor and Sasser scale (1978) was used with grades ranging from 0 to 5, in which: 0 = no egg masses; 1 = presence of 1 to 2 egg masses; 2 = 3 to 10 egg masses; 3 = 11 to 30 egg masses; 4 = 31 to 100 egg masses and 5 = more than 100 egg masses.

The GI was found by counting the number of galls under a light microscope, in the entire root system of each plant/replicate, using a scale from 1 to 5, according to Taylor and Sasser (1978), in which: 0 = absence of galls; 1 = 1 to 2 galls; 2 = 3 to 10 galls; 3 = 11 to 30 galls; 4 = 31 to 100 galls and 5 = more than 100 galls.

The number of eggs and/or J2 per gram of root (NOJ2GR) was obtained by the ratio of total eggs and J2 extracted from the roots and the root mass. The Reproduction Factor (FR) for the different plant species was determined according to Taylor and Sasser (1978). For this, the equation RF = FP / IP was used, where FP = final population and IP = initial population, according to Oostenbrink (1966). IP consisted of the inoculum extracted from tomato roots, quantified and calibrated to contain 5000 eggs and / or juveniles per ml of the suspension. FP was quantified based on J2 present in the soil plus eggs and J2 present in the roots.

2.4.4 Extraction of eggs and J2 from roots

The extraction of eggs and J2 from plant roots was carried out according to the technique adapted from Hussey and Barker (1973) with the roots being cut into pieces of about 1 to 2 cm, crushed with the use of a domestic blender in sodium hypochlorite solution with 0.5% active chlorine, at low speed for 30 s. The crushed was poured into overlaped sieves of 0.355 mm and 0.025 mm. The content of the 0.025 mm sieve was collected with the aid of a pissette. The suspension was cleaned in sucrose solution through the centrifugal flotation technique (JENKINS, 1964). Samples were centrifuged at 650 g for 5 min and the supernatant was discarded. Afterwards, sucrose solution was added, at a concentration of 454 g.L-1, and the sample was centrifuged for 1 min. at 650 g. The suspension was poured through a 0.025 mm sieve and collected in a Becker (maximum 40 ml) with the aid of a pissete.

Aliquots of 150 cm3 of soil were homogenized in plastic container with tap water, stirred for 15 s and poured into overlaped sieves of 0.355 mm and 0.020 mm. The content of the 0.020 mm sieve was collected with the aid of a pissette and the sample was cleaned by the Jenkins technique (1964).

After processing the soil and the roots, eggs and J2 were determined from readings in triplicate, using a Peters’ slide, with the aid of a light microscope.

2.4.5 Classification of plant-species based on the reproductive rate of nematodes

The plant-species were classified into different groups, according to the reproductive rate of the nematodes, as follows: a) Hosts: Average of nematodes with RF>1.0, presence of many galls on the roots, cracks and egg masses (Figure 1A); b) Potential hosts: RF>1.0 in at least one of the replicates, but with an average of RF with few galls or cracks and few egg masses on the roots (Figure 1B); c) Non-hosts: RF = 0, without apparent symptoms and absence or occasional induction of egg masses and/or galls (Figure 1C).

Figure 1 – Root system of plant species classified as host, potential host and non-host of Meloidogyne spp.: A - Triplaris gardneriana, host; B - Copaifera langsdorffii, potential host; C - Terminalia tomentosum, non-host

Figura 1 – Sistema radicular de espécies vegetais classificadas como hospedeira, hospedeira potencial e não hospedeira de Meloidogyne spp.: A – Triplaris gardneriana, hospedeira; B - Copaifera langsdorffii, hospedeira potencial; C - Terminalia tomentosum, não hospedeira

A

B

HOSPEDEIRA

HOSPEDEIRA POTENCIAL

C

NÃO HOSPEDEIRA

Source: Authors (2019)

2.5 Identification of Meloidogyne species by the esterase phenotype

Milky-white females were extracted from tomato roots under a stereomicroscope, placed in 0.5 ml microtubes containing extraction buffer solution (20% sucrose, 2% Triton X-100, 0.01% bromophenol blue and 78% distilled water) and kept on ice. 10 µl of each sample were placed, with the aid of a micropipette, in wells of 8% polyacrylamide gel. The standard sample consisted of Meloidogyne javanica females with a J3 phenotype, as described by Alonso et al. (1999). The race was conducted in a Vertical Electrophoresis System LCV 10X10 (Loccus Biotechnology) for 1 hour at 200 volts, with the tank being kept refrigerated at 4 ºC. After the run, the gel was immersed in a developer solution for esterase (100 mL of 0.05M potassium phosphate buffer pH 6.0, 100 mg of Fast Blue RR Salt and 4.5 mL of 1% alpha-naphthylacetate) and maintained at 37 ºC for 30 minutes. Then, the gel was washed in distilled water and transferred to a fixative solution (45% methanol, 9% acetic acid and 45% distilled water), where it remained for 20 minutes in an oven at 37 ºC. The enzymatic phenotypes were determined based on the number, position and intensity of the bands (ESBENSHADE; TRIANTAPHYLLOU, 1985; 1990).

3 RESULTS AND DISCUSSION

3.1 Nematological analysis of soil, physical-chemical analysis of the substrate and confirmation of the identity of Meloidogyne spp.

Nematological analysis of soil collected from the rizosphere of tillers of the Andropogon bicornis grasses confirmed the absence of Meloidogyne J2 forms. Also, the analysis of tiller roots of the Andropogon bicornis grasses indicated the absence of egg masses. The Sandy texture of the substrate used in the first assay had a clay - silt-sandy ratio of 26% - 7% - 67% and in the second assay 21% - 5% - 74%. These data confirm that the texture of the substrate favored soil movement and infection of Meloidogyne spp. in the inoculated plants (RINALDI; NUNES; MONTECELLI, 2014).

According to the chemical analysis, the substrate used for the greenhouse tests showed low fertility (Table 2) and cover fertilizations with 2 g of N-P-K 4-14-8/pot were used. The Meloidogyne species showed the following phenotypes for the isoenzyme esterase: Meloidogyne javanica (EST-J3), Meloidogyne incognita (EST-I1), Meloidogyne morocciensis (EST-A3) and Meloidogyne paranaensis (EST-P1).

Table 2 – Chemical characterization of substrates used in greenhouse tests

Tabela 2 – Caracterização química dos substratos utilizados nos ensaios em casa de vegetação

Sample

cmolc.dm-3

mg.dm-3

 

 

 

g.dm-3

 

Ca2+

Mg2+

Al3+

H+Al

K+

P (Mehlich)

Zn+

CTC

(T)

V (%)

m (%)

MO

pH CaCl2

Subst.1

1,7

0,2

0

1,3

0,1

11,6

4,1

3,31

60,7

0

10

5,7

Subst.2

1,7

0,2

0

1

0,1

7,7

5,2

2,97

66,3

0

8

6,1

Source: Authors (2019)

Where: 1Substrate used in the 1st test in a greenhouse; 2Substrate used to repeat the test in a greenhouse. Where: Ca2 + - exchangeable calcium; Al3 +: Exchangeable aluminum; K+ - Exchangeable potassium; H + Al - Potential acidity; P (Mehlich - Phosphorus extracted by the Mehlich method; Zn2+: Exchangeable zinc; CTC (t) - Total cation exchange capacity; Mg2 + - Exchangeable magnesium; m (%) - Saturation by aluminum; pH CaCl2 - pH in calcium chloride; MO - Organic matter.

3.2 Reaction of cerrado plants to Meloidogyne spp.

There was variation between the tests regarding the plant response to Meloidogyne spp. The reaction of inoculated plants was more conclusive in the first experiment than in the second. Tomato plants used as susceptibility pattern showed RFs>1.0 for the three Meloidogyne species tested but also with higher reprocutive rates in the first experiment (Table 3).

Table 3 – Averages of Reproduction Factor (RF), Egg Mass Index (EMI), Gall Index (GI) and Number of Eggs and J2 per Root Grass (NEJ2 / GR) in plant species behaved as hosts or potential hosts after four months from inoculation with 5,000 eggs and eventual J2 of Meloidogyne spp.

Tabela 3 – Médias de Fator de Reprodução (FR), Índice de Massas de ovos (IMO), Índice de Galhas (IG) e Número de Ovos e/ou J2 por Grama de Raiz (NOJ2/GR), em espécies vegetais que se comportaram como hospedeiras ou hospedeiras potenciais, após quatro meses da inoculação com 5.000 ovos e/ou J2 de Meloidogyne spp.

Species

Experiment 1

Experiment 2

EMI

GI

NEJ2/GR

RF

EMI

GI

NEJ2/GR

RF

Meloidogyne javanica

Andropogon bicornis

1,2

0

857,6

7,21

1,4

0

120,9

0,67

Esenbeckia leiocarpa

1

0,4

55,72

0,23

0,8

0,2

177,4

0,73

Triplaris gardneriana

2,6

2,2

156,4

2,93

0

0

0

0

Solanum lycopersicum

5

5

1740

5,02

5

5

988

4,3

Meloidogyne morocciensis

Copaifera langsdorffii

0,6

0,2

0,87

0,26

0,6

0,6

0,88

0,004

Triplaris gardneriana

3

1,8

159,7

1,79

0,8

1

4,84

0,05

Solanum lycopersicum

5

5

10677

33,59

5

5

7032

10,59

Meloidogyne incognita

Andropogon bicornis

1,6

0

543,4

1,58

2,8

0

95,59

0,42

Copaifera langsdorffii

1,2

1,8

1772

3,15

0

0

18,05

0,05

Guibourtia hymenifolia

1,4

1,6

291

0,64

0

0

0

0

Triplaris gardneriana

2,6

3,8

365,2

5,97

0

0

5,58

0,08

Solanum lycopersicum

5

5

24760

76,86

5

5

947,2

6,95

Source: Authors (2019)

Abbreviations: RF = Reproduction Factor; EMI = Egg mass index; GI = Gall index; NEJ2/GR = Number of eggs and J2 per gram of root.

The evaluated species that behaved as non-hosts were: Albizia niopoides, Anadenanthera macrocarpa, Anacardium humili, Astronium fraxinifolium, Cybistax antisyphilitica, Dalbergia miscolobium, Jacaranda mimosifolia, Echinolaena inflexa, Elionurus muticus, Eriochrysis filiformis, Eriotheca pubescens, Eugenia tomentosa, Calophyllum brasiliense, Hymenaea stilbocarpa, Inga fagifolia, Inga heterophylla, Kielmeyera coriaceae, Kielmeyera speciosa, Myracrodruon urundeuva, Paspalum plicatulum, Physocalymma scaberrimum, Stryphnodendron adstringens, Handroanthus caraíba, Handroanthus chrysotrichus, Handroanthus impetiginosus, Handroanthus serratifolius, Pseudobombax tomentosum and Terminalia tomentosum.

Plant species that behaved as potential hosts were Copaifera langsdorffii for Meloidogyne morocciensis (FR = 1.15 and FR = 3.20), Esenbeckia leiocarpa for Meloidogyne javanica (FR = 1.1) and Guibourtia hymenifolia for Meloidogyne incognita (FR = 2.26). Andropogon bicornis reacted as host to Meloidogyne javanica and Meloidogyne incognita; Copaifera langsdorffii as host of Meloidogyne incognita and Triplaris gardneriana as host of Meloidogyne javanica, Meloidogyne incognita and Meloidogyne morocciensis.

For most inoculated plant species, the average of reproduction factors produced by Meloidogyne spp. was low, which supports the hypothesis of genetic resistance of the plant species to the nematodes. The non-uniform reaction of replicates, classified as hosts and potential hosts to Meloidogyne spp., supports the hypothesis of intraspecific genetic variability in the cerrado plants tested in this work. This hypothesis gains strength when analyzing individual reproduction factors and not on average. As an example, we have Triplaris gardneriana inoculated with Meloidogyne javanica in experiment 1. The five replicates presented RFs that varied from 0.5 to 11.36, obtaining an average RF = 2.93. Andropogon bicornis also inoculated with Meloidogyne javanica showed an average of RF = 7.21 and a variation of RF from 0 to 35. For Meloidogyne morocciensis, there is Triplaris gardneriana with RF ranging from 0.50 to 2.65 and an average of RF = 1.79. Meloidogyne incognita inoculated on Andropogon bicornis produced RFs ranging from 0 to 5.0 and RF average = 1.58; Copaifera langsdorffii with RF ranging from 0 to 11.80 and RF average of 3.15, as well as Triplaris gardneriana with RF ranging from 0.20 to 7.77 and RF average of 5.97.

Of the plant species studied, Triplaris gardneriana was exclusive in allowing the reproduction of Meloidogyne javanica, Meloidogyne incognita and Meloidogyne morocciensis at mean rates of RF> 1.0. Andropogon bicornis is host of Meloidogyne javanica and Meloidogyne incognita, and Copaifera langsdorffii is host of Meloidogyne incognita. Meloidogyne paranaensis, a nematode species not yet detected in native cerrado, did not show reproductive rates that demonstrated population increase or even maintenance of the population in the inoculated plants. Meloidogyne incognita was the nematode species with the highest number of host plants with an average of RF> 1.0 for three hosts, followed by Meloidogyne javanica with two hosts and Meloidogyne morocciensis with one host plant.

3.3 Symptoms on the roots of plants inoculated with Meloidogyne spp.

The symptoms produced varied according to the inoculated plant species (Figure 2). Of the host-plant species, Andropogon bicornis did not show gall formation and egg masses very evident on the root system (Figure 2A). Copaifera langsdorffii showed cracks with egg masses on the roots, but without evident galls (Figure 2B). Triplaris gardneriana presented evidente galls of different sizes and egg masses (Figure 2D).

Of the potential hosts, Esenbeckia leiocarpa presented egg masses only in newer lateral roots, without gall formation (Figure 2C). Guibourtia hymenifolia presented necrosis and small and barely visible galls on lateral roots, with egg mass formation.

Figure 2 – Symptoms induced by Meloidogyne spp. on the roots of host-plants and potential host-plants from Cerrado: A - Andropogon bicornis (arrow indicating egg mass); B - Copaifera langsdorffii (arrow indicating crack); C - Esenbeckia leiocarpa (arrow indicating swelling with egg mass); D - Triplaris gardneriana (arrow indicating gall with egg mass)

Figura 2 – Sintomatologia em raízes de plantas hospedeiras e plantas hospedeiras potenciais de Meloidogyne spp. do cerrado: A - Andropogon bicornis (seta indicando massa de ovos); B - Copaifera langsdorffii (seta indicando rachadura); C - Esenbeckia leiocarpa (seta indicando intumescimento com massa de ovos); D - Triplaris gardneriana (seta indicando galha com massa de ovos)

A

B

C

D

Source: Authors (2019)

The results obtained in this study revealed new hosts for the tested Meloidogyne species. In the case of a sedentary endoparasite nematode, its survival is related to parasitism and reproduction in plants.

Andropogon bicornis L., a species popularly known as ‘capim-rabo-de-burro’, allowed the reproduction of Meloidogyne incognita and Meloidogyne javanica in its root system, having been classified as host of these nematodes and, being this, the first report in literature. Previously, Café Filho and Huang (1988) reported Pratylenchus brachyurus in association with Andropogon bicornis roots in the cerrado.

In addition to Andropogon bicornis, other grasses were tested in this study for Meloidogyne spp. such as Paspalum pilosum, Paspalum plicatulum, Eriochrysis filiformis, Elionurus muticus and Echinolaena inflexa. These grasses did not allow the reproduction of Meloidogyne spp., having been classified as non-hosts.

The nematodes Meloidogyne javanica and Meloidogyne incognita were reported by Silva et al. (2013) in a cerrado soil sample collected in the Clean Field phytophysiognomy, an area with a predominance of grasses, among which are the species tested in this study. Considering that Meloidogyne javanica and Meloidogyne incognita need host-plants for their survival, it is likely to infer that, in the phytophysiognomies of Cerrado where Andropogon bicornis occurs, both nematodes will infect and reproduce in this host.

Triplaris gardneriana is a plant species adapted to wetlands and commonly known as ‘pau-de-formiga’ or ‘Pajeú’, being indicated for reforestation of riparian forests (LACERDA; BARBOSA; BARBOSA, 2007). In the cerrado, it is found in the phytophysiognomies ‘Cerradão’ and gallery forests (IMAÑA-ENCINAS; MACEDO; PAULA, 2007). This study demonstrated that this species is a new host for Meloidogyne javanica, Meloidogyne incognita and Meloidogyne morocciensis, having no report in the literature of nematodes associated with this plant species. Meloidogyne incognita and Meloidogyne javanica were previously reported by Silva et al. (2013) from soil collected in gallery forest, being an indication that Triplaris gardneriana may host these nematodes in this type of vegetation. Another plant species present in gallery forests is Copaifera langsdorffii (Leguminosae, Caesalpinoideae). This tree has demonstrated the potential for the maintenance and reproduction of Meloidogyne incognita populations in the cerrado.

The ‘ipê’ species such as Handroanthus impetiginosus, Handroanthus serratifolius and Handroanthus caraiba were classified as non-hosts for the nematodes tested in this study. The data obtained by Silva et al. (2013), corroborate with those found in this study where Handroanthus impetiginosus did not allow the reproduction of Meloidogyne javanica in its root system after artificial inoculation.

Despite the negative reaction of the ‘ipê’ species tested for Meloidogyne javanica, Mendes and Cardoso (1978) reported Meloidogyne javanica infecting Handroanthus serratifolius (sin. Tabebuia serratifolia - yellow ipe). Later, Alonso et al. (1999) studied the virulence and aggressiveness of two isolates of Meloidogyne javanica on Handroanthus serratifolius, being one isolate from seedlings of yellow ‘ipê’ and the other from a different host. The authors reported that both isolates had the same reproductive rate on ‘ipê’ (FR = 2.49).

According to Alonso et al. (1999), Handroanthus serratifolius developed the following symptoms: stunting, giant cells in the protoxylem and protofloem, internal egg masses and extensive necrosis in the root cortex. In this study, it was not observed reduction in growth, but well-developed galls and necrosis, with few egg masses.

Natural infection of Handroanthus impetiginosus and Handroanthus serratifolius by Meloidogyne incognita was reported by Antes et al. (2012), in seedlings obtained from a commercial nursery in Western Paraná, Brazil. However, the same authors reported no infection in healthy seedlings of the same plant species when the natural population of Meloidogyne incognita was artificially inoculated, which suggests the occurrence of genetic variability in those plant species.

Handroanthus impetiginosus was also reported as host to Meloidogyne arenaria by Ferreira (1989). The same plant species was studied by Oliveira et al. (1995), who reported the damage caused by Meloidogyne arenaria and Meloidogyne javanica under different inoculum densities. Infected plants produced galls on the roots and a low reproductive rate of the nematode without plant-growth reduction for the different inoculum densities tested. According to Oliveira et al. (1995), the highest values of RF were RF = 0.61 for Meloidogyne javanica with inoculation of 9,000 eggs/J2 and RF = 2.2 for M. arenaria with inoculation of 3,000 eggs/J2.

Further investigation is necessary to comprove the status of Esenbeckia leiocarpa, Guibourtia hymenifolia, and Copaifera langsdorffii, suggested here as potential hosts of Meloidogyne javanica, Meloidogyne incognita, and Meloidogyne morocciensis, respectively.

The conflicting results reported in the literature and related to the reproductive rate of nematodes on plants might be caused by the intraspecific variability found in populations of plants and nematodes (ANTES et al., 2012) and by differences in the methodology used for running experiments such as inoculum density (OLIVEIRA et al., 1995) and its origin (ALONSO et al., 1999 ).

4 CONCLUSION

Triplaris gardneriana was classified as host of Meloidogyne incognita, Meloidogyne javanica and Meloidogyne morocciensis; Andropogon bicornis as host of Meloidogyne javanica and Meloidogyne incognita, and Copaifera langsdorffii as host of Meloidogyne incognita. As potential host-plants were identified: Esenbeckia leiocarpa for Meloidogyne javanica; Guibourtia hymenifolia for Meloidogyne incógnita, and Copaifera langsdorffii for Meloidogyne morocciensis. The remaining plant species were considered non-hosts for the three Meloidogyne isolates. There was no reproduction of Meloidogyne paranaensis on the plants used in this study, which were classified as non-hosts to this nematode. The symptoms observed on the plant roots after inoculation with Meloidogyne spp. were necrosis, cracks, galls, swelling and egg mass production.

ACKNOWLEDGMENTS

The authors thank the National National Council for Scientific and Technological Development (CNPq) and the Coordination for the Improvement of Higher Education Personnel (CAPES) for the scholarships granted to carry out this research. Acknowledgments also to the University of Brasília for the facilities provided to the development of this work and to the Urbanization Company of the New Capital of Brazil (NOVACAP) for donating seeds and plant-seedlings of cerrado to this research.

REFERENCES

ABAD, P.; GOUZY J.; AURY, J. M.; CASTAGNONE-SERENO, P. et al. Genome sequence of the metazoan plant-parasitic nematode Meloidogyne incognita. Nature Biotechnology, Reino Unido, v. 26, p. 909-915. 2008.

ALONSO, S. K.; ALFENAS, A. C. Isoenzimas na taxonomia e na genética de fitonematoides. In: Eletroforese de isoenzimas e proteínas afins, fundamentos e aplicações em plantas e microorganismos, Alfenas AC (Ed). Viçosa, UFV, 1998. 574 p.

ALONSO, S. K.; SANTOS, J. M.; ALFENAS, A. C. FERRAZ, S. Patogênese de Meloidogyne javanica (Treub) Chitwood ao Ipê-Amarelo, Tabebuia serratifolia Nichous. Revista Árvore, Viçosa-MG, v. 23, p. 479-485. 1999.

ANTES, V. A.; COMERLATO, A. P.; SCHUELTER A. R.; CARNEIRO, R. M. D. G.; FURTANETTO, C. Native-plant hosts of Meloidogyne spp. from Western Paraná, Brazil. Brazilian Archives of Biology and Technology, Curitiba-PR, v. 55, p.213-219.2012.

CAFÉ-FILHO, A. C.; HUANG, C. S. Nematóides do gênero Pratylenchus no Brasil. Fitopatologia Brasileira, Brasília-DF, v. 13, p. 232-235, 1988.

DICKSON, D. W.; STRUBLE, F. B. A sieving-staining technique for extraction of egg mass of Meloidogyne incognita from soil. Phytopathology, United States, v. 55, p. 497, 1965.

ESBENSHADE, P.R. & TRIANTAPHYLLOU, A.C. Use of enzyme phenotypes for identification of Meloidogyne species (Nematoda: Tylenchida). Journal of Nematology, United States, v. 17, p. 6-20, 1985.

ESBENSHADE, P.R. & TRIANTAPHYLLOU, A.C. Isozyme phenotypes for the identification of Meloidogyne species. Journal of Nematology, United States, v. 22, p. 10-15, 1990.

FERREIRA, F. A. Doenças florestais no Brasil causadas por nematoides, bactérias, vírus, viróides, micoplasmas e espiroplasmas. In: Ferreira A, editor. Patologia Florestal. Viçosa: UFV, 1989, 570 p.

HUSSEY R. S.; BARKER, K. R. A comparison of methods colleting inocula of Meloidogyne spp. including a new technique. Plant Disease Reporter, United States, v. 57, p. 1025-1028, 1973.

IMAÑA-ENCINAS, J.; MACEDO, L. A.; PAULA, J. E. Florística e fitossociologia de um trecho da floresta estacional semidecidual na área do ecomuseu do cerrado, em Pirenópolis-Goiás. Cerne, Lavras-MG, v. 13, p. 308-320, 2007.

JENKINS, W. R. A rapid centrifugal - flotation technique for separating nematodes from soil. Plant Diasease Reporter, United States, v. 48, p. 692,1964.

LACERDA, A. V.; BARBOSA, F. M.; BARBOSA, M. R. V. Estudo do componente arbustivo-arbóreo de matas ciliares na bacia do rio taperoá, semi-árido paraibano: uma perspectiva para a sustentabilidade dos recursos naturais. Oecologia Brasiliensis, Rio de Janeiro-RJ, v. 11, p. 331-340,2007.

MENDES, B. V.; CARDOSO, C. O. N. Algumas observações histopatológicas de raízes de ipê roxo parasitadas por Meloidogyne arenaria. Summa Phytopathologica, Jaboticabal-SP, v. 4, p. 49-54, 1978.

OLIVEIRA, C. M. G.; GOULART, A. M. C.; MONTEIRO, A. R.; FERRAZ, L. C. C. B. Suscetibilidade e danos causados em ipê-roxo por Meloidogyne javanica e M. arenaria. Nematologia Brasileira, Piracicaba-SP, v.19, p. 89-92, 1995.

ONKENDI, E. M.; KARIUKI G. M.; MARAIS, M. MOLELEKI, L. N. The threat of root-knot nematodes (Meloidogyne spp.) in Africa: a review. Plant Pathology, Reino Unido, v. 63, p.727-737, 2014.

OOSTENBRINK, M. (1966) Major characteristics of the relation between nematodes and plants. Mededelingen van de Landbouwhogeschool, Wageningen-Holanda, v. 66, n. 1, p. 46, 1966.

RINALDI, L. K.; NUNES, J. MONTECELLI, T. D. N. Efeito de texturas do solo sobre populações de Meloidogyne javanica e Meloidogyne incognita em soja. Cultivando o Saber, Paraná, v. 7, p. 83-101, 2014.

SILVA, G. S; SANTOS, J. M.; FERRAZ, S. Novo método de coloração de ootecas de Meloidogyne sp. In: Congresso Brasileiro de Nematologia, XII. Anais. Dourados, MS, 1988.

SILVA J. G. P.; FURLANETTO, C.; ALMEIDA, M. R. A.; ROCHA, D. B.; MATTOS, V. S.; CORREA, V. R.; CARNEIRO, R. M. D. G. (2013) Ocorrence of Meloidogyne spp. in Cerrado vegetations and reaction of native plants to Meloidogyne javanica. Journal of Phytopathology, Germany, v. 162, p. 449-455, 2013.

SOUZA, R. M.; DOLINSKI, C. M.; HUANG, S. P. Survey of Meloidogyne spp. in native cerrado of Distrito Federal, Brazil. Fitopatologia Brasileira, Brasília-DF, v. 19, p. 463-465, 1994.

TAYLOR, A. L.; SASSER, J. N. Biology, identification and control of root-knot nematodes (Meloigyne spp.). Raleight- North Caroline State University, 1978, 111 p.

Authorship Contribution

1 – Reinaldo Rodrigues Pimentel

Investigation, Methodology, Writing – original draft, Writing – review & editing

2 – Ramon Lira dos Anjos

Data curation, Investigation, Methodology, Validation

3 – Adriana Andrade Ferreira

Data curation, Investigation, Methodology, Validation

4 – Gisele Pereira Domiciano

Conceptualization, Formal Analysis, Investigation, Methodology, Supervision

5 – Regina Célia de Oliveira

Resources, Supervision, Methodology

6 – Cleber Furlanetto

Project administration, Supervision, Writing – original draft, Writing – review & editing