Characterization and levels of resistance in Coffea arabica ×Coffea racemosa hybrids to Leucoptera coffeella

doi:10.21203/rs.3.rs-4365695/v1

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Characterization and levels of resistance in Coffea arabica ×Coffea racemosa hybrids to Leucoptera coffeella

https://doi.org/10.21203/rs.3.rs-4365695/v1

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Coffee leaf miner (CLM) Leucoptera coffeella stands out as a primary insect pest of arabica coffee plants. Coffee breeding for CLM-resistance has used the species Coffea racemosa as gene donor for C. arabica, resulting in the development of the resistant commercial hybrid ‘Siriema AS1’. However, no previous study has characterized the resistance, and whether there is variation in the levels expressed in progenies of ‘Siriema’ to CLM. This study aimed to characterize the type, by antixenosis or antibiosis, and the levels of resistance in segregating progenies of ‘Siriema’ plants to CLM. Experiments were conducted under laboratory conditions with artificial infestation of CLM adults in oviposition cages, where dual-choice preference assays compared each tested ‘Siriema’ progeny with the susceptible cv. Arara. A follow-up no-choice assay evaluated five selected ‘Siriema’ progenies on the development of CLM compared to ‘Arara’. As main results, ‘Siriema’ progenies were equally susceptible to CLM oviposition, and were overall stimulant relative to cv. Arara. However, there was low CLM larval survival and injury intensity on the selected ‘Siriema’ genotypes, indicating the expression of moderate levels of antibiosis-resistance, while ‘Arara’ and one ‘Siriema’ progeny were moderately susceptible. These findings further our understanding on the type and levels of resistance in ‘Siriema’ genotypes, aiding the development of resistant coffee hybrids and deployment of management strategies to CLM.

Coffee leaf miner

Host plant resistance

Antixenosis

Antibiosis

IPM

Coffee leaf miner (CLM), Leucoptera coffeella, is a key pest of coffee crops
‘Siriema’ hybrid is CLM-resistant, but the resistance has not yet been characterized
Progenies of ‘Siriema’ were equally or more oviposited by CLM than cv. Arara
Most progenies of ‘Siriema’ caused larval mortality and reduced injury of CLM
‘Siriema’ progenies express resistance to CLM with varying levels of antibiosis

Coffee leaf miner (CLM) Leucoptera coffeella (Guérin-Méneville & Perrottet) (Lepidoptera: Lyonetiidae) is recognized as a primary pest in major coffee-producing regions of Brazil (Souza et al. 1998; Almeida et al. 2020), the largest coffee producer and exporter worldwide (EMBRAPA 2022). CLM is a monophagous and holometabolic insect that feeds and reproduces only in the coffee leaves. Injury by CLM to coffee plants is caused by the larvae, which tunnel the leaf tissues of the palisade mesophyll while feeding, promoting reductions in leaf area and photosynthetic capacity, premature leaf fall, thus compromising the production of coffee fruits (Crowe 1964; Parra 1985; Mendonça et al. 2016). CLM is considered a major pest of Coffea arabica because of its widespread occurrence and negative impact on coffee yield (Righi et al. 2013; Almeida et al. 2020).

Chemical control is the most commonly used tactic to reduce CLM populations and damage in regions of high pest infestation. However, the intense use of insecticides causes many negative effects such as the mortality of natural enemies, accumulation of toxic residues in the environment and food, and the selection of resistant pest populations (Guedes et al. 2017; Leite et al. 2020). The emergence of resistant CLM populations has led conventional insecticides such as organophosphates and pyrethroids to become inefficient. Furthermore, repeated insecticide applications for CLM control and the high cost of purchasing modern insecticides elevate the production costs (Leite et al. 2020).

The use of resistant coffee cultivars is one of the most promising alternatives for CLM management, especially in sustainable coffee production systems. Host plant resistance is advantageous because insect pest populations can be reduced below economic injury levels, without causing ecological imbalances and negative effects on beneficial organisms and natural enemies of pests, leaving no toxic residues in the environment as compared to insecticide use. Cultivation of resistant cultivars is also compatible with most control methods of integrated pest management (Smith 2005; Boiça Júnior et al. 2013; Vendramim and Rosales 2019).

Host plant resistance to insects can be expressed as antixenosis, antibiosis, and tolerance (Smith 2005). Antixenosis is related with phenotypic plant traits, including physical, chemical, and morphological features that affect insect behavior and is manifested as feeding and/or oviposition non-preference to that plant genotype. Antibiosis is governed by the presence of plant traits that are unfavorable to insect development; the underlying antibiosis-resistance mechanisms, which can be chemical and/or morphological, acutely or chronically impair insect’s biological parameters such as the survival of immature stages, life cycle duration, weight gain, and the fecundity and fertility of emerged adults. Tolerance, on the other hand, are related to the expression of physiological compensatory mechanisms that make plants to better withstand herbivory, without posing negative effects on insects’ behavior and development, resulting in lower yield losses (Smith and Clement 2012).

Breeding of resistant coffee cultivars have been done through the use of both traditional and molecular selection techniques, using the species C. racemosa as gene donor for C. arabica (Carvalho and Monaco 1968; Guerreiro Filho et al. 1999). Although several studies have focused on the improvement of arabica coffee resistance to CLM, all commercial cultivars exhibited susceptibility. In the last decade, ‘Siriema AS1’ was developed by the Procafé Foundation and released in the marketplace, which is recognized as the only resistant variety to CLM (Carvalho et al. 2013). This variety is also resistant to coffee leaf rust (Hemileia vastatrix Berk & Br.), another important biotic threat to coffee production.

Coffee seedlings of ‘Siriema’ progenies used in this study originated from a cross between C. arabica and C. racemosa, where hybridization was initially done at the Agronomic Institute of Campinas. This genotype is drought-tolerant and resistant to coffee leaf rust, and these characteristics were acquired by crossing with the variety Catimor. This plant population received the name ‘Siriema’, and then it has been improved by the genetic breeding program of the Procafé Foundation, which has made most progress in obtaining materials resistant to CLM, including the commercial hybrid ‘Siriema AS1’ (Carvalho et al. 2013).

Although field evaluations have shown very low infestations of CLM in 'Siriema AS1' plants (Costa 2022), no study has yet characterized the resistance effects on the oviposition behavior and biological development of CLM under controlled conditions. Here we focused on the evaluation of antixenosis and antibiosis categories of resistance because in field observations ‘Siriema’ plants were less infested by CLM, which were unlikely due to tolerance mechanisms. Knowledge on the effects of ‘Siriema’ progenies on CLM ovipositon and development would support genetic breeding programs to obtain Coffea varieties with resistance levels to CLM. Additionally, defining the categories of resistance in ‘Siriema’ plants, either by antixenosis or antibiosis, and the levels of resistance expressed can aid in the deployment of integrated pest management strategies using this cultivar against CLM. Therefore, this study aimed to characterize the type and levels of resistance in segregating progenies of ‘Siriema’ plants (C. racemosa x C. arabica) to CLM in the laboratory.

Obtaining the coffee seedlings of ‘Siriema’ progenies

Coffee seedlings of the ‘Siriema’ progenies tested were obtained from the Procafé Foundation germplasm bank, in Varginha, Minas Gerais state, Brazil. The seedlings were sent to the Department of Entomology of the Federal University of Lavras (UFLA) and kept in a greenhouse free of insects and pathogens. The seedlings used in bioassays were approximately 7 months old. The progenies of ‘Siriema’ plants evaluated in this study are described in Table 1. The commercial cultivar Arara was used as the standard genotype, which is classified as susceptible to CLM infestation (Costa 2022).

Table 1

Evaluated ‘Siriema’ progenies obtained from the Procafé Foundation
Genotype	‘Siriema’ progeny
T70	3–86 (30) (30 − 3)
T71	3–86 (41 − 1)
T72	3–86 (62)
T73	3–86 (63)
T66	3–86 (09)
T67	3–86 (13)
T65	3–86 (06)
T69	3–86 (20)
T68	3–86 (20)

Rearing of coffee leaf miner in the laboratory

The resistance type of ‘Siriema’ progenies was evaluated in the Laboratory of Plant Resistance and Integrated Pest Management at UFLA. The adults of CLM were reared in acrylic cages (100 × 60 × 100 cm), using a methodology similar to that described by Katiyar and Ferrer (1968). The cages were kept in a climatized room in the laboratory (29 ± 3°C, 70 ± 10% RH, and 14L:10D h) to shorten the insect cycle and obtain more individuals. The adults were fed a 10% sucrose solution impregnated in a filter paper that was hung in the upper part of cages. For reproduction of CLM, coffee seedlings of susceptible cultivars were used inside the cages as substrates for adult oviposition and larval development.

Evaluation of antixenosis-resistance in ‘Siriema’ progenies to coffee leaf miner

To evaluate antixenosis-resistance in the laboratory, dual-choice oviposition preference assays were performed, contrasting each tested ‘Siriema’ progeny with ‘Arara’. Completely expanded leaves of the coffee seedlings were cut at the petioles and placed in phenolic foam soaked with water and transferred to plastic cages (60 × 60 × 70 cm). Each cage contained two equidistant phenolic foam stands, each holding five leaves of a tested ‘Siriema’ progeny and five leaves of ‘Arara’. Adults of CLM were collected from the rearing colony using flat-bottom tubes and then released in a proportion of 150 adults per oviposition cage. The experimental design was completely randomized, and six replicates were used for each binary combination, represented by six cages.

Before CLM infestation in the cages, the relative chlorophyll index of the leaves was recorded using the SPAD-502 chlorophyll meter (Konica Minolta Sensing Inc., Tecnal, Piracicaba, Brazil). We used this approach to estimate potential differences in chlorophyll contents between leaves of tested genotypes that could explain CLM oviposition preference (Torres Netto et al. 2005; França and Carvalho 2016). After 72h of insect release in the cages, the leaves were removed and the eggs laid quantified under a stereoscope (40× magnification). After counting the eggs, the leaves were photographed using a digital camera and the images were processed in the ImageJ software (National Institutes of Health, Bethesda, USA). Next, the number of eggs/10 cm² was standardized to minimize the interference of leaf area between coffee genotypes on CLM oviposition.

Based on the number of eggs laid in each genotype, the oviposition preference index (OPI) was determined as follows: OPI = [(T-S)/(T + S)] x 100; where T = number of eggs laid in the assessed ‘Siriema’ genotypes, and S = number of eggs laid on the standard susceptible cv. ‘Arara’. OPI varies from + 100 (stimulant), 0 (neutral) to -100 (deterrent) (Fenemore 1980; Silva et al. 2014). The classification of ‘Siriema’ genotypes based on the oviposition stimulus (stimulant/neutral/deterrent) as compared to ‘Arara’ was done from comparison of the mean numbers of eggs on the treatments with the standard susceptible cultivar, taking into account the mean standard error (± SE).

Evaluation of antibiosis-resistance in ‘Siriema’ progenies to coffee leaf miner

The treatments of this bioassay consisted of the following ‘Siriema’ genotypes based on the results of the preference assay: T66, T68, T69, T70, T71, and ‘Arara’. In this no-choice bioassay, each cage (60 × 60 × 70 cm) received only one coffee genotype. The leaves were placed by the petioles in trays containing moist vermiculite to maintain turgidity. Approximately 150 adults were released in each cage and allowed to oviposit for 48h. Next, the trays with leaves were removed from the cages, and the excess eggs were eliminated using a fine paintbrush, keeping three eggs per leaf. The experimental design was completely randomized, with six treatments and four replicates. The replicates consisted of four trays 15 leaves each, totaling 60 leaves evaluated per treatment.

From the eggs laid on the leaves, the biological cycle of CLM was monitored on different genotypes to record the durations of the egg, larval, and pupal stages, allowing to determine the total duration of the life cycle. After the pupae had developed on the leaves, they were individually stored in flat-bottom tubes (2 x 8 cm) to evaluate the emergence of adults and record their longevity. The mortality of CLM in the larval and pupal stages were also recorded.

After 17 days of CLM oviposition, a qualitative injury score was assigned to the mined leaves, according to an injury scale proposed by Guerreiro Filho (1999) based on the type of lesion: 1 = small punctate lesions; 2 = small filiform lesions; 3 = large irregular lesions; and 4 = large rounded lesions (Fig. 1). Consequently, the ‘Siriema’ progenies were classified according to their levels of antibiosis-resistance to CLM based on the leaf injury scores (Table 2).

Table 2

Injury scores for evaluation of the type of lesions caused by coffee leaf miner and the corresponding resistance levels of coffee genotypes
Injury score	Resistance level	Description
1	Highly resistant	Small punctate lesions
2	Moderately resistant	Small filiform lesions
3	Moderately susceptible	Large irregular lesions
4	Highly susceptible	Large rounded lesions

Statistical analysis

Data obtained in the bioassays were checked for the normality of residuals (Shapiro-Wilk) and homogeneity of variances (Bartlett test). Data of pupal survival, egg stage duration, and injury scores were transformed to arcsin\(\sqrt{x+1}\) to meet the assumptions of ANOVA, and then back-transformed for presentation in tables. When significant difference was obtained by ANOVA, means of treatments were compared by Tukey’s test (α = 0.05). The data of the oviposition preference index (OPI) did not meet the assumptions of normality and homoscedasticity and were subjected to the non-parametric Kruskal-Wallis test. The analyses were performed in the software IBM SPSS Statistics v. 21 (IBM Corp., Armonk, NY, USA).

In the dual-choice oviposition assay, no significant differences were observed among ‘Siriema’ progenies. Based on the calculation of the OPI, the ‘Siriema’ progenies T70, T71, T72, T73, T67, T65, T69, and T68 were classified as stimulants, and the T66 genotype as neutral in the binary comparison with ‘Arara’. None of the evaluated ‘Siriema’ genotypes were classified as deterrent to CLM (Table 3). In addition, when contrasting the mean numbers of eggs cm^− 2 in all ‘Siriema’ progenies with those in cv. Arara, the oviposition rate was twice as much in the genotypes of ‘Siriema’, with significant difference (t_{1, 52} =-4.0; P < 0.001) (Table 3).

Table 3

Mean numbers (± SE) of eggs of *Leucoptera coffeella* per 10-cm² leaf in ‘Siriema’ genotypes and cv. Arara, oviposition preference index (OPI), and classification of the attractiveness stimulus
Genotype		cv. Arara	OPI¹	Classification²
T70	2.1 ± 0.5	1.9 ± 0.4	11.5 ± 27.8 a	Stimulant
T71	2.2 ± 0.4	0.8 ± 0.1	25.0 ± 31.9 a	Stimulant
T72	2.0 ± 0.4	0.8 ± 0.2	38.7 ± 9.5 a	Stimulant
T73	2.8 ± 0.5	1.4 ± 0.3	24.9 ± 18.4 a	Stimulant
T66	1.3 ± 0.2	1.3 ± 0.4	3.2 ± 19.3 a	Neutral
T67	1.6 ± 0.3	1.3 ± 0.2	9.7 ± 6.8 a	Stimulant
T65	1.3 ± 0.2	0.4 ± 0.2	67.7 ± 13.3 a	Stimulant
T69	3.0 ± 0.6	1.1 ± 0.3	25.6 ± 23.6 a	Stimulant
T68	3.7 ± 0.9	1.5 ± 0.4	23.6 ± 17.2 a	Stimulant
Mean	2.2 ± 0.2	1.1 ± 0.1	t = -4.00	P < 0.001
¹Means followed by the same letter in column do not differ by Kruskal-Wallis test (P < 0.05). ²The average standard error (± SE) of the means was used for stimulus classification: SE = ± 6.35. There is significant difference between the progenies of ‘Siriema’ and cv. Arara

There were no significant differences in the mean values of SPAD relative chlorophyll index between the resistant ‘Siriema’ genotypes and the commercial cultivar Arara as measured in the same leaves used in oviposition preference assays. The mean values were similar regardless of the genotype, with values ranging from 51.0 to 58.6 SPAD units (Table 4).

Table 4

Mean values (± SE) of SPAD indices in ‘Siriema’ progenies and cv. Arara
Genotype	Siriema genotype		Arara		P-value
T70	54.1 ± 1.7	a	56.7 ± 1.3	a	0.255
T71	56.0 ± 1.2	a	53.7 ± 1.4	a	0.219
T72	58.6 ± 1.5	a	54.6 ± 1.6	a	0.080
T73	56.2 ± 1.5	a	53.6 ± 1.5	a	0.244
T66	58.5 ± 1.7	a	55.3 ± 1.6	a	0.191
T67	58.6 ± 1.6	a	56.4 ± 1.3	a	0.323
T65	57.6 ± 1.4	a	54.7 ± 1.4	a	0.167
T69	56.0 ± 1.2	a	57.2 ± 1.0	a	0.450
T68	51.0 ± 1.2	a	52.7 ± 1.2	a	0.352
Means followed by the same letter in rows do not differ by t test (P < 0.05)

Based on the results of the oviposition assay, the progenies T66, T68, T69, T70, and T71 were selected and further evaluated for the expression of antibiosis in comparison with ‘Arara’ under no-choice condition. The survival of CLM larvae was significantly lower (F_{5, 23}=19.02; P < 0.001) in the ‘Siriema’ progenies T66, T70, T69, and T71. In these genotypes, CLM larval survival varied between 38.6% (T70 progeny) and 58.4% (T71 progeny). Cultivar Arara and progeny T68 did not differ for the larval survival, and exhibited higher viability rates, being 99% in ‘Arara’ and 97% in T68 (Table 5). No significant difference (F_{5, 23}=2.39; P = 0.078) was observed among coffee genotypes for CLM pupal survival. The percentage of pupal survival varied from 82.8 to 100% among tested genotypes (Table 5).

Table 5

Means (± SE) of survival of the larval and pupal stages of *Leucoptera coffeella* on ‘Siriema’ genotypes and cv. Arara
Genotype	Larval survival (%)		Pupal survival (%)
Arara	99.0 ± 0.9	a	96.4 ± 2.1	a
T68	97.0 ± 3.0	a	98.6 ± 1.3	a
T66	44.5 ± 8.3	b	98.0 ± 1.9	a
T70	38.6 ± 5.2	b	100.0 ± 0.0	a
T69	35.9 ± 7.7	b	100.0 ± 0.0	a
T71	58.4 ± 9.7	b	82.8 ± 9.9	a
P-value	< 0.001		0.078
Means followed by the same letter in columns do not differ by Tukey’s test (P < 0.05).

Regarding the durations of the CLM stages, the egg stage (F_{5, 23}=0.96; P = 0.463) showed no significant differences among coffee genotypes, with mean incubation period of 7.45 days. The durations of the larval (F_{5, 23}=1.65; P = 0.198), pupal (F_{5, 23}=2.43; P = 0.075), and adult (F_{5, 23}=1.37; P = 0.281) stages were also similar among genotypes, with mean durations of 12.41, 6.2, and 9.33 days, respectively. For the total life cycle (F_{5, 23}=0.29; P = 0.137) of CLM, no significant differences were found, with an average duration of 26.0 days (Table 6).

Table 6

Mean (± SE) duration of the egg, larval, pupal, and adult stages and the total life cycle of *Leucoptera coffeella* on ‘Siriema’ progenies and cv. Arara
Genotype	Egg		Larva		Pupa		Adult		Total cycle
Arara	7.3 ± 0.2	a	12.25 ± 0.2	a	6.75 ± 0.2	a	9.25 ± 0.2	a		26.3 ± 0.2	a
T68	7.2 ± 0.1	a	12.25 ± 0.2	a	6.25 ± 0.2	a	9.50 ± 0.5	a		25.7 ± 0.2	a
T66	7.7 ± 0.1	a	12.75 ± 0.2	a	6.25 ± 0.2	a	8.75 ± 0.6	a		26.7 ± 0.4	a
T70	7.5 ± 0.2	a	12.00 ± 0.0	a	6.25 ± 0.2	a	9.25 ± 0.2	a		25.7 ± 0.3	a
T69	7.6 ± 0.1	a	12.50 ± 0.2	a	6.25 ± 0.2	a	9.00 ± 0.4	a		26.3 ± 0.2	a
T71	7.2 ± 0.3	a	12.75 ± 0.2	a	5.50 ± 0.2	a	10.25 ± 0.4	a		25.5 ± 0.3	a
P-value	0.463		0.198		0.075		0.281		0.137
Means followed by the same letter in columns do not differ by Tukey’s test (P < 0.05).

For the evaluation of type and size of injury formed on the leaves after 17 days of CLM larval development, there was significant difference (F_{5, 23}=18.40; P < 0.001) among genotypes. Progeny T68 and ‘Arara’ showed similar injury scores and higher than the other genotypes, presenting predominantly large irregular and large rounded lesions (Table 7). Based on the injury scores, the ‘Siriema’ progenies T66, T69, T70, and T71 were classified as moderately resistant to CLM by antibiosis, whereas the progeny T68 and ‘Arara’ were moderately susceptible.

Table 7

Mean (± SE) injury scores of lesions caused by *Leucoptera coffeella* on ‘Siriema’ genotypes and cv. Arara, and the corresponding antibiosis-resistance level
Genotype	Injury scores		Antibiosis-resistance level
Arara	2.9 ± 0.04	a	Moderately susceptible
T68	3.1 ± 0.08	a	Moderately susceptible
T66	1.8 ± 0.1	b	Moderately resistant
T70	1.5 ± 0.1	b	Moderately resistant
T69	1.6 ± 0.2	b	Moderately resistant
T71	2.0 ± 0.2	b	Moderately resistant
P-value	< 0.001		-
Means followed by the same letter in column do not differ by Tukey’s test (P < 0.05).

This study characterized the type and levels of resistance in plants of ‘Siriema’ progenies to CLM, a primary pest of arabica coffee crops. In dual-choice oviposition assays contrasting each ‘Siriema’ genotype with the commercial cultivar Arara, CLM preferred ‘Siriema’ leaves to lay eggs in most binary comparisons, being these progenies overall classified as stimulants compared to ‘Arara’. Additionally, CLM females laid twice as many eggs when considering the mean oviposition in all ‘Siriema’ progenies compared to cv. Arara. Leaf size, indicated by the numbers of eggs per 10-cm² leaf, and leaf color intensity, measured as the relative chlorophyll index, are not traits underpinning CLM oviposition preference. Therefore, based on the CLM oviposition assays in the laboratory, it became clear that ‘Siriema’ genotypes do not express the resistance by antixenosis. Our study is the first to evince that the resistance in ‘Siriema’ plants is manifested as antibiosis, mainly affecting CLM larval survival and injury intensity.

Plants expressing antixenosis affect the behavior of herbivorous insects during host-plant selection, and is manifested as insect non-preference to oviposit on those plants. On the other hand, antibiosis negatively affects biological aspects of insect development (Painter 1951; Smith 2005; Boiça Júnior et al. 2013). The low larval survival and small punctuate and filiform lesions of CLM on the ‘Siriema’ genotypes indicate the expression of antibiosis-resistance. According to Smith (2005), when antibiosis is expressed, insect mortality or delay in its life cycle can be due to chemical substances harmful to insect development, causing high mortality rates in the immature stages, weight reduction, deformities, and lowering of adults’ reproductive potential.

Although there are no studies in the literature on the effects of ‘Siriema’ genotypes on CLM oviposition, previous work has been done with its progenitors. Matos et al. (2011) studied the preference of CLM in genotypes of C. arabica, C. congensis, C. canephora, and C. racemosa. As results, seedlings of the C. arabica cultivars ‘Mundo Novo IAC-515-20’, ‘Tupi IAC 1669-33’, ‘Icatu Precoce IAC 3282’, and ‘Catuaí Amarelo IAC 62’ were preferred when compared with C. canephora cv. Apoatã IAC 2258, while the latter were more oviposited than C. racemosa. Similar results were found when the authors assayed detached leaves, being C. congensis intermediately preferred than C. arabica and C. canephora. In their study, C. racemosa was the least preferred by CLM, but in none of the assays was the cultivar Arara used. The level of attractiveness of ‘Arara’ to CLM adults when compared with other coffee genotypes is scarcely known. Aparecida and Fernandes (2021) found that ‘Arara’ had lower CLM infestation than ‘Catuaí’, ‘Catucaí’, and ‘Mundo Novo’ in the field.

According to Cardenas (1981) and Matos et al. (2011), green color intensity may influence CLM oviposition preference such that darker leaves were preferred over light-green leaves, which may explain the lower oviposition in C. canephora. In our study, it became clear that there was no antixenosis-resistance expression in the ‘Siriema’ genotypes; on the contrary, nearly all ‘Siriema’ genotypes were classified as stimulants to CLM oviposition in comparison to ‘Arara’. There was either no difference in the relative chlorophyll index between leaves of ‘Siriema’ progenies and ‘Arara’. The SPAD values of coffee leaves are highly correlated with total nitrogen (N) content and constitutes a tool for diagnosing photosynthetic system integrity (Torres Netto et al. 2005; França and Carvalho 2016). In previous studies, high leaf N levels and more intense green color were correlated with higher attraction of CLM adults (Matos et al. 2011; Theodoro et al. 2014; Sabino et al. 2018). Because the ‘Siriema’ progenies and cv. Arara did not differ in the SPAD values, CLM preference for ‘Siriema’ genotypes should be due to plant traits other than leaf color intensity, e.g. the release of volatile compounds attractive to the adults. In addition to volatiles, Santiago-Salazar et al. (2021) suggested that leaves’ cuticle waxes and texture may influence CLM oviposition preference.

Given the results obtained in the no-choice assay, an antibiosis-related mechanism to CLM is suggested in the resistant ‘Siriema’ progenies. The expression of this resistance category is indicated because there was low larval survival and most injury lesions were classified as small punctate and filiform (injury scores 1 and 2), which are characteristic of high and moderate levels of antibiosis-resistance, respectively. Similar results were found by Guerreiro Filho et al. (1999) and Ramiro et al. (2004) for the classification of CLM lesions in other coffee genotypes. According to Guerreiro Filho et al. (1991), variation in lesions of CLM among the coffee species C. racemosa, C. stenophylla, and C. kapakata is probably due to the action of phytochemical compounds that interfere with larval development.

The small lesions in the ‘Siriema’ resistant progenies indicated that CLM larvae initiated feeding on the leaf mesophyll, but the presence of deterrent, antinutritional and/or toxic compounds, or even morphological features, hindered larval feeding and growth, resulting in less severe lesions and higher larval mortality. In some leaf replicates specifically in the ‘Siriema’ T66 progeny, there was evidence that the effects on CLM also occurred in the egg stage (Fig. 2). However, because we did not record the viability of eggs, we cannot account for the proportion of CLM individuals that died in the egg or larval stage in the T66 progeny. In the other tested ‘Siriema’ genotypes, there was no signal of egg mortality, and all the negative effects on CLM survival was observed after the larvae started tunneling the leaf mesophyll tissue.

The main characteristics studied in resistant coffee species were leaf anatomy, leaf morphology, phenological development, and phytochemical compounds (Guerreiro Filho 2006). Ramiro et al. (2004) observed anatomical leaf characteristics of the parental species C. arabica and C. racemosa, and in resistant and susceptible hybrids from this cross, and correlated with CLM-resistance. Although leaf tissue thickness differed between the coffee species, it did not between resistant and susceptible hybrids, suggesting the evaluated traits are not involved in the resistance. Medina Filho et al. (1977) evaluated several cultivars, hybrids, and backcrosses of Coffea species for CLM-resistance under field conditions; they found no differences in the infestation of plants with different ploidy degrees, leaf thickness or leaf size, and concluded that these traits have no relationship with CLM-resistance. Magalhães et al. (2008) found a positive correlation of foliar caffeine levels and CLM oviposition; although caffeine is not a volatile compound, its effect on egg laying was aided by the volatile p-cymene.

Pupal viability of CLM was not affected by the resistant genotypes in our study, despite the observed high larval mortality. The CLM individuals that could develop on the resistant genotypes may have accumulated enough energy to molt into pupae. This suggests that there was no chronic toxicity that compromised the further stages of CLM development. According to Rodriguez and Vendramim (1996), the negative effects of host plants on insect survival in function of toxic compounds is more significant in the larval stage, because the larvae ingest the phytochemicals. The durations of egg, larval, pupal, and adult stages of CLM were statistically similar between the resistant and susceptible genotypes in our study. One possible hypothesis is that the surviving CLM larvae became healthy pupae through compensatory mechanisms such as food retention in the gut, and in some cases by lengthening the larval stage to obtain nutrient and energy reserves necessary to accomplish metamorphosis (Reynolds et al. 1985).

Although resistant coffee genotypes showed high mortality of CLM larvae, the females did not detect differences in the host plants upon oviposition. According to the preference-performance hypothesis, insect females should prefer to lay eggs on host plants that increase the performance and survival of their offspring (Jaenick 1978; Gripenberg et al. 2010). According to Renwick and Chew (1994), polyphagous insects have a greater opportunity to choose among potential plant hosts that offer high-quality food; on the other hand, monophagous insects, such as CLM, have limited options for host-plant choice. In some cases, female's choice is not always beneficial for the offspring. In the study of Santiago-Salazar et al. (2021), there were no differences in CLM oviposition in a multiple-choice assay that could correlate with the distinct larval performance, so that the authors suggested that complementary studies should be conducted to assess other factors on the CLM preference-performance relationship.

In a hypothesis formulated by Harvey and Fortuna (2012), invasive plants may affect the ability of herbivorous insects to find potential host plants by producing attractive odors that serve as a trap for the insects, when in fact the introduced plant has antinutritional properties. Thus, a possible hypothesis for the contrasting results found in our study between CLM females choice and larval development in ‘Siriema’ plants is the similarity between C. arabica x C. racemosa hybrids with C. arabica susceptible cultivars, which may produce volatiles attractive to CLM or the leaf surface is suitable for oviposition; however, the composition of the leaf mesophyll tissues of ‘Siriema’ is not suitable to sustain the larval feeding and development. Therefore, in addition to being cultivated as resistant hybrids in the field, the ‘Siriema’ genotypes could serve as trap plants for CLM in the borders of crops grown with other cultivars, which would not sustain larval development, lowering the pest population densities. This management strategy is encouraged to be evaluated in field conditions for validation.

Another interesting result found in our study specifically in the ‘Siriema’ progeny T66 was that some leaf replicates killed CLM in the egg stage. This was only observed in the Siriema T66 genotype, and the necrosis and egg desiccation effects resembled a hypersensitive response that is manifested against pathogens (Fig. 2). Unfortunately, it is not possible to account for the proportion of CLM individuals that died in the egg or larval stage as we did not record the eggs viability.

Hypersensitive responses (HR) are programmed cell death induced by bursts of reactive oxygen species that impair pathogen spread and colonization in host plants (Hilker and Fatouros 2016). There is accumulated evidence that some genotypes of plants species can express induced resistance against insect eggs after recognizing egg-associated molecular patterns, including indoles, benzyl cyanide, bruchins, and phosphatidilcholynes (Hilker and Fatouros 2015, 2016; Bertea et al. 2020; Stahl et al. 2023; Lortzing et al. 2024). Although HR-like against insect eggs were reported in plants of the Brassicacea, Solanaceae, and Ulmaceae plants, no information has yet been found in coffee plants showing induced-resistance responses against CLM eggs. According to Lortzing et al. (2024), egg deposition can lead to hydrogen peroxide and salicylic acid accumulation at the oviposition site, which can elicit HR-like symptoms. Therefore, further research should focus on the CLM egg-induced responses in the T66 genotype and other segregating progenies of ‘Siriema’, as well as on the underlying mechanisms to introgress the resistance genes into commercial coffee hybrids.

As main conclusions, the ‘Siriema’ progenies (C. arabica x C. racemosa) tested in this study do not affect the oviposition of CLM, and thus these plants do not express antixenosis-resistance. The low survival rates in the larval stage of CLM and the presence of small punctuate and filiform foliar lesions indicate that the moderate resistance levels expressed in the ‘Siriema’ progenies are through antibiosis.

Acknowledgments

The authors thank the Coordination for the Improvement of Higher Education Personnel (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES), the National Council for Scientific and Technological Development (Conselho Nacional de Desenvolvimento Científico e Tecnológico - CNPq), the Minas Gerais Research Foundation (Fundação de Amparo à Pesquisa do Estado de Minas Gerais - FAPEMIG), the Procafé Foundation, and the Federal University of Lavras (Universidade Federal de Lavras).

Author contributions

DCM conducted the experiment, data analysis, and manuscript draft; BHSS designed the experiment, analyzed data, reviewed and edited the manuscript; CHSC bred the coffee progenies and supplied seedlings; OGF conceptualized the study and provided research information.

Funding

This study was funded by the MCTIC/CNPq Universal CNPq Edital Nº 28/2018 (Process no. 436302/2018-7); by the Coordination for the Improvement of Higher Education Personnel (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES); by the National Council for Scientific and Technological Development (Conselho Nacional de Desenvolvimento Científico e Tecnológico - CNPq); and by the Minas Gerais Research Foundation (Fundação de Amparo à Pesquisa do Estado de Minas Gerais - FAPEMIG).

Conflict of interest

The authors declare no conflict of interest.

Ethical approval

It is not applicable.

Consent to participate

It is not applicable.

Consent to publish

It is not applicable.

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Characterization and levels of resistance in Coffea arabica ×Coffea racemosa hybrids to Leucoptera coffeella

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Abstract

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Key Messages

Introduction

Materials and methods

Obtaining the coffee seedlings of ‘Siriema’ progenies

Rearing of coffee leaf miner in the laboratory

Evaluation of antixenosis-resistance in ‘Siriema’ progenies to coffee leaf miner

Evaluation of antibiosis-resistance in ‘Siriema’ progenies to coffee leaf miner

Statistical analysis

Results

Discussion

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References

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