Assessment of Methods and Criteria for Screening Psidium spp. for Resistance to Meloidogyne enterolobii

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Nematologia Brasileira 211

Assessment of Methods and Criteria for Screening Psidium spp. for

Resistance to Meloidogyne enterolobii

Guilherme B. Miranda1_{, Ricardo Moreira Souza}1*_{& Alexandre P. Viana}2

1_{Laboratório de Entomologia e Fitopatologia, Universidade Estadual do Norte Fluminense Darcy Ribeiro, Av. Alberto} Lamego 2000, 28015-620 Campos dos Goytacazes (RJ) Brazil.

2_{Laboratório de Melhoramento Genético Vegetal, Universidade Estadual do Norte Fluminense Darcy Ribeiro.} *_{Corresponding author: ricmsouza@censanet.com.br}

Recebido para publicação em 10 / 09 / 2010. Aceito em 22 / 02 / 2011 Editado por Guilherme L. Asmus

Summary - Miranda, G.B., R.M. Souza & A.P. Viana. 2010. Assessment of methods and criteria for screening

Psidium spp. for resistance to Meloidogyne enterolobii.

This study aimed to improve the methodology for screening guava and “araçá” genotypes for resistance to

M. enterolobii. Guava seedlings ‘Paluma’ were inoculated with 500 eggs, and 135 days later their entire or half

root system (cut along its longitudinal axis) was processed for egg extraction and estimation of the final nematode population (Pf). The Pf counts were compared through F test, which confirmed (P < 0.05) that genotypes can be evaluated for resistance to M. enterolobii by processing just half of the root system, thereby allowing the resistant plants to be replanted, cloned and further studied. In another experiment, 22 guava genotypes and four “araçá” genotypes were inoculated and processed as described above for comparison of three criteria routinely used in screenings to classify genotypes: reproduction factor (RF) sensu Oostenbrink (1966), RF sensu Moura & Régis (1987) and statistical grouping through Scott-Knott test. The results showed the first criterion to be the most appropriate. An ANOVA of the Pf counts of all genotypes revealed significant differences between plants of the same genotype. Further studies are in progress to detect the source of this intra-genotype variation, which could be either genetic variation within the open-pollinated genotypes of guava and “araçá” or an error intrinsic to screening tests, or both.

Key words: guava root-knot nematode, guava decline, guava, “araçá”.

Resumo - Miranda, G.B., R.M. Souza & A.P. Viana. 2010. Avaliação de métodos e critérios para seleção de genótipos de Psidium spp. para resistência a M. enterolobii.

Neste trabalho objetivou-se aprimorar a metodologia para seleção (screening) de genótipos de goiabeira e araçazeiro resistentes a M. enterolobii. Mudas de goiabeira ‘Paluma’ foram inoculadas com 500 ovos e após 135 dias tiveram todo ou somente metade do sistema radicular (cortado em seu eixo longitudinal) processado para extração de ovos e estimativa da população final do nematoide (Pf). As contagens da Pf foram comparadas por meio do teste F, o qual confirmou (P < 0.05) que genótipos podem ser avaliados para resistência a M.

enterolobii processando-se somente metade do sistema radicular das plantas, o que permite o replantio, clonagem

e estudos futuros das plantas resistentes. Em outro experimento, 22 genótipos de goiabeira e quatro genótipos de araçazeiro foram inoculados e avaliados como descrito acima para a comparação dos seguintes critérios de classificação de genótipos quanto à resistência: fator de reprodução (FR) sensu Oostenbrink (1966), FR sensu Moura & Régis (1987) e agrupamento por meio de Scott-Knott. A análise dos resultados demonstrou que o primeiro critério é o mais apropriado. Considerando-se o conjunto de dados de Pf dos 26 genótipos testados, a ANOVA detectou diferenças estatísticas significativas entre plantas de mesmo genótipo. Estudos estão sendo conduzidos para se determinar a fonte desta variação intra-genotípica, que pode ser devida à polinização

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Introduction

Meloidogyne enterolobii Yang & Eisenback, 1983

(junior synonym M. mayaguensis Hammah & Hirschmann, 1988) is an emerging threat to tropical agriculture (Rodríguez et al., 2007). Its distribution includes a number of countries, in which it has been related to yield losses in crops such as guava (Psidium

guajava L.), Caribbean cherry, coffee, tomato and

soybean, among others. In field surveys and experimental settings, M. enterolobii has been found to reproduce on many plant species of several botanic families, including tomato, pepper, sweet potato, cowpea and soybean that bear Meloidogyne spp.-resistance genes (Fargette, 1987; Fargette et al., 1996 cited by Cetintas et al., 2008; Brito et al., 2007a,b; Carneiro et al., 2007; Cetintas et al., 2008).

In Brazil, Gomes et al. (2011) reported that M.

enterolobii predisposes guava trees to extensive root

decay caused by Fusarium solani, resulting in a complex disease named “guava decline”. A subsequent study (unpublished) involving root samples from different Brazilian regions confirmed the nationwide incidence of this disease, which has caused widespread decimation of orchards and a direct economic impact estimated at over R$ 112 million as of 2008 (Pereira

et al., 2009). Although Gomes et al. (2010)

demonstrated the potential of managing guava decline with the use of organic soil amendments, nematode resistance is considered the best control strategy, because nematode-free guava trees are imune to root decay caused by F. solani.

There have been various screenings of guava and “araçá” (Psidium spp. or Eugenia spp.) genotypes – which are phylogenetically related - for resistance to

M. enterolobii, M. acrita (syn. M. incognita), M. arenaria, M. incognita, M. javanica and M. hapla (Cuadra &

Quincosa, 1982; Casassa et al., 1997; Maranhão et al., 2001, 2003; Burla et al., 2007; Carneiro et al., 2007; Milan, 2007; Almeida et al., 2009; Scherer, 2009). In these studies, the authors used a wide range of methodology and also different criteria to classify the

genotypes as nematode resistant or susceptible. There has been only one study aiming to standardize the methodological procedures for such screenings (Burla et al., 2010). Standardization is essential to allow comparisons between screenings conducted at different times and by different research groups (Hussey & Janssen, 2002). For M. enterolobii, Burla et al. (2010) indicated that an inoculum level of 500 or 2,000 eggs per plant is well suited for genotype screenings, instead of the higher inoculum levels - as high as 15,000 eggs / plant – used in some studies. The authors also suggested that genotype screenings should be evaluated 135-180 days after inoculation, and that the variables final nematode population (Pf

= eggs + J₂ extracted from the root systems) and

reproduction factor (RF= Pf / inoculum) were better than Pf / gram of root to assign host suitability.

This study reports further methodological evaluations: upon inoculation of 26 guava and “araçá” genotypes, it was assessed whether Pf counts obtained from processing half the plant root system was statistically equivalent to counts obtained from processing the entire root system. Since guava and “araçá” plants generally survive the trimming of their roots and replanting, obtaining reliable Pf and RF data from half root systems is advantageous. It allows individual resistant plants to be kept alive for further studies, an interesting approach for open-pollinated species such as guava and “araçá”.

It was also assessed whether RF should be used to classify the genotypes as proposed by Oostenbrink (1966) (RF < 1 = resistant; RF > 1 = susceptible) or by Moura & Régis (1987) (without a specific RF cutoff value). In the latter sense, the genotype with highest RF is taken as the susceptible standard, and the other genotypes are classified according to their ability to reduce the highest RF: 0-25 % reduction = highly susceptible; 26-50 % = susceptible; 51-75 % = weakly resistant; 76-95 % = moderately resistant; 96-99 % = resistant; 100 % = highly resistant or immune. These two criteria, which have been used by different authors aberta de goiabeiras e araçazeiros, ou a um erro metodológico intrínseco aos testes de screenings, ou a ambos. Palavras-chaves: nematoide-das-galhas da goiabeira, declínio da goiabeira, goiaba, araçá.

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to screen guava and “araçá” genotypes, were compared with the statistical analysis of Pf counts through F and Scott-Knott tests.

Typically, genotypes are classified as resistant or susceptible according to the average response observed from the five to ten replicates (plants) of each genotype that were inoculated. For open-pollinated plants such as guava and “araçá”, this may not be the best approach when one uses seedlings produced from true seeds, since unknown resistance genes may flow across the plant population and vary in the progenies. In the present study, Pf counts of each plant of each genotype were analyzed to assess whether resistance should be evaluated for each plant individually, as opposed to the standard approach described above.

Material and Methods

General procedures. Twenty-two guava genotypes and four “araçá” genotypes (P. guineense

Swartz or P. cattleyanum Sabine) were used in this study (Table 1). For all guava genotypes, the seedlings were produced from 7-10 cm-long stem cuttings. Whenever the guava genotype seemed somewhat variable phenotypically, e.g. in its leaf or fruit morphology, the cuttings were obtained from a single tree. Because the stem cuttings of “araçá” did not emit roots, even with the use of hormones, these genotypes had their seedlings produced from true seeds. The seedlings were produced in 500 ml plastic bags filled with the growth

substrate Plantmax®_.

For the first and second experiments (see below), seedlings at the stage of four pairs of leaves were individually transplanted to 3,000-cm3_{plastic bags filled}

with a mixture of riverbed sand, soil and cattle manure (matured) at a 2:1:1 ratio. Each seedling was inoculated with 10 ml of a suspension calibrated to 500 eggs of

M. enterolobii, which was poured into four holes around

the seedling collar. Seedlings were maintained in a greenhouse with mean daily, mean maximum and Table 1 - Genotypes of cultivated or wild guava (Psidium guajava) and wild “araçá” (P. guineense or P. cattleyanum) collected in Rio de

Janeiro State, Brazil, and evaluated for resistance to Meloidogyne enterolobii.

1_{Genotype numbers refer to the collection of the Nematology Research Group at Universidade Estadual do Norte Fluminense Darcy Ribeiro.}

Genotype number1 _{/ identification} _{Collecting site (municipality) / GPS coordinates} _{Type of seedling used}

136 / guava ‘Paluma’ Bom Jesus do Itabapoana, lat. 21o _{9’44"S; long. 41}o_35’55"W _{Cuttings from different trees (C, Dt)}

93 / guava ‘Pedro Sato II’ Cachoeira de Macacu, lat. 22o_{34’37"S; long. 42}o_43’12"W _{C, Dt}

94 / guava ‘Hitigio’ Cachoeira de Macacu, lat. 22o_{34’39"S; long. 42}o_43’10"W _{C, Dt}

95 / guava ‘Tsumori’ Cachoeira de Macacu, lat. 22o_{34’39"S; long. 42}o_43’9"W _{C, Dt}

39 / guava ‘Sassaoka’ Bom Jesus do Itabapoana, lat. 21o_{9’7"S; long. 41}o_37’5"W _{C, Dt}

41 / wild guava Bom Jesus do Itabapoana, lat. 21o_{9’7"S; long. 41}o_37’5"W _{C, Dt}

36 / guava ‘Vita I’ Bom Jesus do Itabapoana, lat. 21o_{9’7"S; long. 41}o_37’5"W _{C, Dt}

40 / guava ‘Pedro Sato I’ Bom Jesus do Itabapoana, lat. 21o_{9’7"S; long. 41}o_37’5"W _{C, Dt}

35 / guava ‘Século XXI’ Bom Jesus do Itabapoana, lat. 21o_{9’7"S; long. 41}o_37’5"W _{C, Dt}

135 / guava ‘Rica’ Bom Jesus do Itabapoana, lat. 21o_{9’44"S; long. 41}o_35’55"W _{C, Dt}

134 / guava ‘Kumagai Branca’ Bom Jesus do Itabapoana, lat. 21o_{9’44"S; long. 41}o_35’55"W _{C, Dt}

84 / wild guava São João da Barra, lat. 21o_{39’42"S; long. 41}o_26’41"W _{Cuttings from a single tree (C, St)}

85 / wild guava São João da Barra, lat. 21o_{39’42"S; long. 41}o_26’41"W _{C, St}

87 / wild guava São João da Barra, lat. 21o_{39’21"S; long. 41}o_2’7"W _{C, St}

56 / wild guava Cachoeira de Macacu, lat. 22o_{31’22"S; long. 42}o_41’49"W _{C, St}

117 / wild “araçá” (P. cattleyanum) São João da Barra, lat. 21o_{41’22"S; long. 41}o_3’20"W _{True seeds}

115 / wild “araçá” (P. cattleyanum) Campos dos Goytacazes, lat. 21o_{45’47"S; long. 41}o_19’2"W _{True seeds}

116 / wild “araçá” (P. cattleyanum) Campos dos Goytacazes, lat. 21o_{45’41"S; long. 41}o_18’30"W _{True seeds}

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mean minimum temperatures of 30.5, 37.9 and 23.0

o_{C, respectively. Seedlings were irrigated and fertilized}

as needed.

One hundred and thirty-five days after inoculation (dai), the plant root systems were washed free of soil and individually processed for egg extraction according to Hicks & Simmons (2003) with modifications: the root systems were individually placed in 1-l glass vials filled with 500 ml of 6 %

aqueous solution of Qboa®_{commercial bleach}

(approximately 5.25 % sodium hypochlorite). The vials

were shaken in a commercial shaker (TECNAL®_,

model TE240) for 4 min. at 130 cyles per min. The resulting suspension was poured onto 100 and 500 mesh sieves, and three 1 ml aliquotes were examined to obtain nematode Pf counts.

First experiment. In order to evaluate whether processing half or entire root systems for egg extraction was statistically equivalent, 14 guava seedlings ‘Paluma’ were inoculated with M. enterolobii as described above, and maintained in a greenhouse in an entirely randomized arrangement. Upon evaluation (at 135 dai), the entire root systems of seven plants were processed for egg extraction as described above. The root systems of the other seven plants were cut in half along its longitudinal axis, and the roots were processed as described above. These plants, with the remaining roots, were immediately replanted. The Pf counts obtained from half root systems were multiplied by two, and all counts (non-transformed) were compared through F test.

Second experiment. In order to compare different criteria to classify the genotypes as nematode resistant or susceptible, five to seven seedlings of each of the genotypes listed in Table 1 were inoculated as described above and maintained in a greenhouse in an entirely randomized arrangement. Since extracting eggs from half or entire root systems was statistically equivalent (see results), at 135 dai half root systems of the plants were processed for egg extraction as described above. The Pf counts (non-transformed) were compared through F and Scott-Knott tests. The genotype groups generated by the Scott-Knott test were compared with the groups obtained when the criteria proposed by Oostenbrink (1966) and Moura & Régis (1987) were applied.

To assess the variation between the plants of each genotype, for each 1 ml suspension aliquote of each individual plant of each genotype, the Pf counts (non-transformed) were analyzed using a model with random blocks and plant variation inside the genotype / block, to determine the variation between genotypes and between plants of each genotype. The model used

was Yijk = bj + gi + pk / gi + eijk (NID,0,σ2_{), in}

which bj= effect of block, gi= effect of genotype, pk/gi= effect of plant within the genotype, and eijk= experimental error. The data were analyzed using ANOVA, and the variance was estimated for plants within genotypes and between genotypes. The data were analysed using the software Genes (Cruz, 2006).

Results and Discussion

Processing whole vs. half root system. The F test of Pf counts indicated (F < 0.05) that screenings of Psidium spp. can be conducted through processing of just half the plant root system if it is cut carefully along its longitudinal axis (Table 2; Figure 1). This non-destructive method allowed the replanted plants to grow normally. This half root system approach was used to process the 26 guava and “araçá” genotypes examined in the following experiment (see below), with all the plants of all genotypes growing well after replanting.

Comparison of different criteria to classify genotypes. Based on the Pf counts, the Scott-Knott test created (F < 0.01) two groups of genotypes, one of them comprised of the cultivated guavas ‘Hitigio’ and ‘Tsumori’, the wild guavas 87, 98, 101, 102 and 108, and the “araçás” 115, 116 and 117 (Table 3).

Based on the Oostenbrink’s classification scheme, the only resistant genotypes were “araçás” 115, 116 and 117, which presented RF< 1 (Table 3). The genotypes grouped with these “araçás” through the Scott-Knott test presented RFs varying from 11.2 through 23.3, which suggest that Scott-Knott is not useful to indicate which genotypes should be used in a breeding or grafting program. Likewise, Moura & Régis’ scheme classified the wild guava 98 as moderately resistant (it presented a RF = 11.2), and genotypes classified as weakly resistant had RFs as high as 23.3. The flaw in Moura & Régis’ scheme lies in classifying the genotypes based on a relative RF: a

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Table 2 - F test for final nematode population (Pf) in plants of guava (Psidium guajava) ‘Paluma’ inoculated with Meloidogyne enterolobii

and evaluated 135 days later by processing whole or half root systems.

1_{Values (not transformed) are average of seven replicates (plants) per treatment. Values followed by the same letter in the column are}

statistically equivalent at F < 0.05.

Treatments Pf1 _{Calculated F value} _{Tabled F value} _{CV %}

Whole root system 62,171A 0.031 4.28 34.8

Half root system 64,228A - -

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Table 3 - Genotypes of cultivated or wild guava (Psidium guajava) and wild “araçá” (P. guineense or P. cattleyanum) inoculated with

Meloidogyne enterolobii in greenhouse and evaluated 135 days later for final nematode population (Pf) and reproduction factor (RF =

Pf / 500).

1_{Values are average of five to seven replicates (plants) per genotype.} 2_{Scott-Knott grouping at F < 0.01.}

3,4_{Genotype classification regarding resistance to M. enterolobii according to Oostenbrink (1966) and Moura & Régis (1987): HS = highly}

susceptible; S = susceptible; WR = weakly resistant; MR = moderately resistant; R = resistant.

Genotype number Pf(x 1,000)1 _{Scott-Knott groups} _RF _Oostenbrink _{Moura & Régis}

/ identification based on Pf2 ₍₁₉₆₆₎3 ₍₁₉₈₇₎4

136 / guava ‘Paluma’ 22.4 A 44.8 S HS

93 / guava ‘Pedro Sato II’ 16 A 31.9 S S

94 / guava ‘Hitigio’ 8.1 B 16.3 S WR

95 / guava ‘Tsumori’ 10.9 B 21.8 S WR

39 / guava ‘Sassaoka’ 15.4 A 30.8 S S

41 / wild guava 17.7 A 35.3 S S

36 / guava ‘Vita I’ 21 A 42 S HS

109 / wild guava 14.5 A 28.9 S S

40 / guava ‘Pedro Sato I’ 22.7 A 45.3 S HS

35 / guava ‘Século XXI’ 18.1 A 36.2 S S

135 / guava ‘Rica’ 24.6 A 49.3 S HS

108 / wild guava 10.5 B 21 S WR

134 / guava ‘Kumagai Branca’ 17.3 A 34.6 S S

84 / wild guava 13.9 A 27.8 S S 85 / wild guava 26.5 A 52.9 S HS 87 / wild guava 11.7 B 23.3 S WR 56 / wild guava 22 A 44.1 S HS 51 / wild guava 14.7 A 29.5 S S 98 / wild guava 5.6 B 11.2 S MR 99 / wild guava 24.3 A 48.7 S HS 101 / wild guava 7.2 B 14.4 S WR 102 / wild guava 10.5 B 21.1 S WR

117 / wild “araçá” (P. cattleyanum) 0.19 B 0.38 R R

111 / wild “araçá” (P. guineense) 20.5 A 41.1 S HS

highly susceptible standard genotype will drag all the other genotypes up the resistance scale, leading the nematologist or breeder to classify as resistant some genotypes with fairly high RFs.

Based on this study, it seems clear that Ostenbrink’s RF is better than Scott-Knott test and Moura & Régis’ scheme to identify guava and “araçá” genotypes useful for breeding and grafting programs. Nonetheless, genotypes with RF just above 1 – susceptible in a strict sense - may be useful for further crossings and field trials. In the field, genotypes with RF just above 1 would be likely to sustain slow growth of a M.

enterolobii population over the years in a guava

plantation; however, it could be possible that the plants would not suffer a heavy burden on their physiology, therefore remaining resistant to the main pathogen

involved in this pathosystem, F. solani.

Assessment of variation between plants of each genotype. Interestingly, despite the uniformity of the experimental conditions, for each genotype the Pf counts showed a C.V. % varying from 25 to 171 % (results not shown), which suggests that the host response varied considerably among the plants of each genotype. The ANOVA conducted considering the Pf counts of each 1 ml aliquote of each plant of each genotype confirmed (P < 0.05) the differences between the plants, which were numerically expressed

by s_w2_{= 53.24 (Table 4). Even within genotypes that}

were considered susceptible to M. enterolobii (RF > 1) (Table 3), there were individual plants whose RF was below or just above 1 (Table 5). These individual plants will be cloned and rescreened to assess their resistance

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to M. enterolobii.

It is hypothesized that the variation observed in host response between plants of the same genotype

may stem from two sources: i) experimental error(s) intrinsic to nematode resistance screenings, which might explain the variation observed in wild guava Table 4 – ANOVA and estimates of variance between genotypes (σg2) and between plants of the same genotypes (sw2) in genotypes of cultivated or wild guava (Psidium guajava) and wild “araçá” (P. guineense or P. cattleyanum) inoculated with Meloidogyne enterolobii in greenhouse and evaluated 135 days later for final nematode population.

Source DF Mean Square Variance component

Block 3

Genotype 25 3151025.8 σw2 + kσ2 +jkσg2

Residual 339 78028.9 σw2 + kσ2

Plants within genotypes 143 4232782.9 σw2

Average 14,646.3 σg2 = 39.38 —

C.V. % 12.27 σw2 = 53.24 —

Table 5 - Genotypes and plants of cultivated or wild guava (Psidium guajava) and wild “araçá” (P. cattleyanum) inoculated with

Meloidogyne enterolobii in greenhouse and evaluated 135 days later for final nematode population (Pf) and reproduction factor (RF =

Pf / 500).

1_{Values are average of three counts performed on three 1 ml aliquotes per plant.}

2_{Genotype classification of resistance to M. enterolobii according to Oostenbrink (1966): R= resistant; S= susceptible.} 3_{“*” indicates plants that, although strictly susceptible, will be retested.}

Genotype number Plant number Pf1 _RF _{Classification}2,3

/ identification 117 / wild “araçá” 1 0 0 R 117 / wild “araçá” 2 26 0.1 R 117 / wild “araçá” 3 80 0.2 R 117 / wild “araçá” 4 106 0.2 R 117 / wild “araçá” 5 906 1.8 S* 117 / wild “araçá” 6 133 0.3 R 117 / wild “araçá” 7 80 0.2 R 115 / wild “araçá” 1 320 0.6 R 115 / wild “araçá” 2 160 0.3 R 115 / wild “araçá” 3 133 0.3 R 115 / wild “araçá” 4 80 0.2 R 115 / wild “araçá” 5 186 0.4 R 115 / wild “araçá” 6 26 0.1 R 115 / wild “araçá” 7 453 0.9 R 116 / wild “araçá” 1 0 0 R 116 / wild “araçá” 2 186 0.4 R 116 / wild “araçá” 3 320 0.6 R 116 / wild “araçá” 4 133 0.3 R 116 / wild “araçá” 5 160 0.3 R 116 / wild “araçá” 6 53 0.1 R 116 / wild “araçá” 7 266 0.5 R

35 / guava ‘Século XXI’ 2 266 0.5 R

56 / wild guava 1 213 0.4 R 98 / wild guava 3 1,280 2.6 S* 94 / guava ‘Hitigio’ 3 400 0.8 R 84 / wild guava 4 400 0.8 R 84 / wild guava 5 346 0.7 R 51 / wild guava 1 560 1.1 S* 109 / wild guava 1 453 0.9 R 87 / wild guava 2 1,093 2.2 S* 87 / wild guava 3 373 0.7 R 135 / guava ‘Rica’ 3 1,280 2.6 S*

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genotypes 51, 56, 84, 87 and 98, of which the stem cuttings used to produce the seedlings were collected from single trees, and ii) genetic variation, which might explain the variation observed in wild guava 109 and in ‘Hitigio’, ‘Rica’ and ‘Século XXI’, of which the stem cuttings used to produce the seedlings were collected from a population of trees. In Brazil, it has been noticed that guava cultivars are somewhat variable phenotypically, and ‘Hitigio’ has not been officially characterized. Further experiments have been set up to test those hypotheses.

The results of the present study suggest that for under-studied plant species such as guava and “araçá”, there is a need to understand the sources of errors in genotype screenings for resistance to M. enterolobii. Proper protocols will be crucial once research advances beyond screenings, which have been mostly unsuccessful in finding resistance sources, into a phase of breeding guava cultivars through classical or molecular-assisted methods.

Acknowledgments

The authors are indebted to Viveiro Itamudas Ltda for providing genotypes to be screened and access to its seedling-producing facility, and to Dr. Marcelo Souza (Museu Nacional, Universidade Federal do Rio de Janeiro) for the taxonomic identification of the “araçás”.

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