agronomy

Article
Breeding Peaches for Brown Rot Resistance in Embrapa
Maximiliano Dini 1,2, * , Maria do Carmo Bassols Raseira 3 , Silvia Scariotto 4 and Bernardo Ueno 3

1 Programa de Pós-Graduação em Agronomia, Faculdade de Agronomia Eliseu Maciel,

Universidade Federal de Pelotas, Capão do Leão 96010-900, Rio Grande do Sul, Brazil
2 Instituto Nacional de Investigación Agropecuaria (INIA), Estación Experimental INIA Las Brujas,
Ruta 48 km 10, Rincón del Colorado, Canelones 90100, Uruguay
3 Embrapa Clima Temperado, Empresa Brasileira de Pesquisa Agropecuária, BR 392, km 78,
Pelotas 96010-971, Rio Grande do Sul, Brazil
4 Campus Pato Branco, Universidade Tecnológica Federal do Paraná, Via do Conhecimento, km 1,
Pato Branco 85503-390, Paraná, Brazil
* Correspondence: [email protected]; Tel.: +598-96256299

Abstract: Brown rot, caused by Monilinia spp., is the main stone fruit disease. Major efforts to detect
sources of resistance are being applied by several breeding programs worldwide. The main objective
of this study was to seek sources of brown rot resistance, as well as to study the segregation, estimate
the heritability, verify the possible existence of the maternal effect, and estimate the genetic advances.
For this purpose, 20 parents and 303 seedlings, representing 16 breeding families, and ‘Bolinha’
(control) have been phenotyped for fruit reaction to brown rot using wounded and non-wounded
inoculation procedures in 2015–2016, 2016–2017, and 2017–2018 growing seasons. Wounded fruits
were very susceptible to brown rot incidence, however, the incidence and severity of non-wounded
fruits showed high variability among the evaluated genotypes. Conserva 947 and Conserva 1600
and their progeny, had lower disease incidence and severity than most of the evaluated genotypes.
Genetic gain estimation was −5.2 to −30.2% (wounded fruits) and between −15.0 to −25.0% (non-
wounded fruits) for brown rot resistance. Selected genotypes were equal to or better than ‘Bolinha’
Citation: Dini, M.; Raseira, M.d.C.B.; in relation to brown rot resistance, with several of them far superior in fruit quality than ‘Bolinha’,
Scariotto, S.; Ueno, B. Breeding
demonstrating the progress of the Embrapa Peach Breeding Program.
Peaches for Brown Rot Resistance in
Embrapa. Agronomy 2022, 12, 2306.
Keywords: Prunus persica (L.) Batsch; Monilinia fructicola (Winter) Honey; genetic resistance; progeny
https://doi.org/10.3390/
segregation; genetic advance; lesion; sporulation
agronomy12102306

Academic Editor:
Bénédicte Quilot-Turion

Received: 31 July 2022

1. Introduction
Accepted: 12 September 2022 Peach (Prunus persica L. Batsch) breeding programs started in Brazil, in the early 1950s,
Published: 26 September 2022 in the Instituto Agronomico de Campinas, São Paulo State. A few years later a similar
program started in the Rio Grande do Sul State. The latter was first coordinated by the
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
Agricultural Secretary of Rio Grande do Sul. However, in the early 1960s, the program
published maps and institutional affil-
was moved to Pelotas (Rio Grande do Sul), and then coordinated by a federal institution.
iations.
With the advent of Empresa Brasileira de Pesquisa Agropecuária (Embrapa) in 1973, the
program was not only preserved but also increased [1].
The Rio Grande do Sul State is the main peach producer, with about 64% of the
Brazilian production, occupying more than 11,4 thousand hectares, in the year 2019. Pelotas,
Copyright: © 2022 by the authors. where the Embrapa Peach Breeding is located, stands out as the largest national producer
Licensee MDPI, Basel, Switzerland. of peaches, accounting for more than 11% of the country’s total production [2]. The area is
This article is an open access article characterized by very humid weather and mild temperatures, so very favorable to fungus
distributed under the terms and diseases. Among them, brown rot, caused by Monilinia fructicola (Winter) Honey, is by far
conditions of the Creative Commons the most important disease and for that reason, one of the top priorities of the Embrapa
Attribution (CC BY) license (https:// Peach Breeding Program is the search for M. fructicola resistance [1]. Under the above
creativecommons.org/licenses/by/ mentioned conditions, the disease symptoms may be visible in 48 h after infection. Brown
4.0/).

Agronomy 2022, 12, 2306. https://doi.org/10.3390/agronomy12102306 https://www.mdpi.com/journal/agronomy

Agronomy 2022, 12, 2306 2 of 13

rot damages can occur from flowering to post-harvest. The main symptoms are blossom
blight, cankers in branches, and brown rot lesions in fruits [3–5].
The use of genetic resistance has been limited in commercial orchards, since com-
mercial peach cultivars immune to brown rot are still unavailable. However, there are
significant differences in susceptibility among available genotypes [3]. Brown rot control
is mainly done by fungicide sprays [6]. Thus, genetic resistance is a priority in many
breeding programs worldwide, mainly due to the emergence of fungus isolates resistant to
fungicides [7–11] and the increase in environmental concerns and workers’ and consumers’
health [1,12,13].
The Brazilian cultivar Bolinha shows higher levels of resistance in fruits than most
cultivars, however, its poor fruit quality, together with problems of premature fruit fall,
discourage its commercial cultivation [14–18].
Fruit resistance to brown rot is quantitative and polygenic trait [13,19,20]. This type
of resistance slows the development of the epidemic in orchards, in spite of the genotype
being susceptible to the disease [21,22].
Knowledge of the genetic, phenotypic, and environmental parameters that directly
or indirectly influence the characters of economic importance in a crop is fundamental
for breeding program guidance [23]. Given the limited information on peach brown rot
resistance in Brazilian genotypes and with the intention of contributing to the genetic
improvement of this crop, the objectives of this study were to evaluate the distribution
of brown rot resistance in fruits of different populations; verify the possible existence of
maternal effect for this character; estimate the heritability; identify genotypes with higher
levels of resistance; estimate the genetic advance for this character in the Embrapa Peach
Breeding Program.

2. Materials and Methods

2.1. Plant Material
The study was developed at Embrapa Clima Temperado, in Pelotas, Rio Grande do
Sul, Brazil (Lat. 31◦ 400 S, Long. 52◦ 260 W, alt. 57 m asl.) in the 2015–2016, 2016–2017 and
2017–2018 seasons. The fruit reaction to brown rot was tested in 16 F1 progenies of the
Embrapa Peach Breeding Program, including ten progenies from five reciprocal crosses.
Fruits of individual seedlings and their parents were evaluated (Table 1).

Table 1. Progeny identification, parents, and number of seedlings of each progeny, in the Peach
Breeding Program at Embrapa Clima Temperado, Pelotas, Rio Grande do Sul, Brazil.

Progeny Parent ♀ Parent ♂ N◦ Seedlings

2008.159 Conserva 1526 ‘Cerrito’ 7
2009.38 ‘Cerrito’ Conserva 1526 23
2012.26 Cascata 1055 ‘Chimarrita’ 18
2012.43 ‘Chimarrita’ Cascata 1055 25
2012.49 Conserva 672 Conserva 1526 18
2012.61 Conserva 1526 Conserva 672 7
2012.52 Conserva 947 Conserva 1600 17
2012.66 Conserva 1600 Conserva 947 12
2012.68 Conserva 1662 ‘Maciel’ 24
2012.88 ‘Maciel’ Conserva 1662 17
2012.31 Cascata 1359 Cascata 1577 31
2012.46 Chorão ‘Maciel’ 25
2012.99 Necta 506 ‘Sunmist’ 20
2012.107 Necta 532 Necta 480 25
2012.111 Necta 540 ‘Morena’ 25
2012.114 ‘Rubimel’ TX2D163 21
Agronomy 2022, 12, 2306 3 of 13

Seedlings were planted spaced 0.5 m apart on the row and 5 m between rows. The
parent trees were planted in triplicate in a work collection of Embrapa, spaced 2 m between
trees and 5 m between rows.
A total of 303 seedlings, 20 parents (cultivars and advanced selections), and the cultivar
Bolinha were evaluated for their reaction to brown rot on W and NW fruits.

2.2. Experimental Design and Treatments

The experimental design was completely randomized, and each genotype was con-
sidered one treatment. Five wounded (W) (2015–2016, 2016–2017, and 2017–2018 seasons)
and five non-wounded (NW) fruits (2016–2017 and 2017–2018 seasons) per seedling were
inoculated. In the same seasons, 10 inoculated fruits (five W and five NW), per each one of
the three parents clones were evaluated. The Bolinha cultivar was used as a standard for
low brown rot susceptibility [14,15].

2.3. Pathogen Culture, Conidia Production, and Inoculation

The procedure adopted for inoculations was the same described by Dini et al. [24].
Briefly, the fungus isolate was obtained from four peach orchards in Embrapa Clima
Temperado. For the wounded inoculations (penetration of 1 mm into the fruits) a 100 µL
syringe coupled to a repeating dispenser 50× (Hamilton® ) was used. In both treatments
(W and NW) 10 µL suspension of M. fructicola (2.5 × 104 conidia mL−1 ) and Tween-80®
(0.1 g L−1 ) was used, followed by incubation at 23 ± 1 ◦ C and 12 h photoperiod for three
days [16,18,19,24–27].

2.4. Brown Rot Evaluation

Incidence and severity 72 h after inoculation (HAI) were evaluated according to
Dini et al. [24]. Brown rot incidence (BRI) and sporulation presence (SPP) were calculated
as the percentage of fruits that presented symptoms and sporulation of M. fructicola, re-
spectively; lesion diameter (LD) and sporulation diameter (SPD) were evaluated through
two perpendicular measurements per fruit in the equatorial region; lesion area (LA) and
lesion sporulation (SPA) were calculated and expressed as the percentage of fruit that was
affected by the lesion and sporulation, respectively. Thus, fruit size was considered in each
measurement making them comparable, which was important since fruit size was very
variable among genotypes.

2.5. Statistical and Genetic Analysis

To evaluate the progeny segregation and to test for the possibility of maternal effect,
relative frequency histograms were constructed with the severity data. The maternal effect
was tested by comparing the F1 progeny with the F1 reciprocal progeny, by the t-test
(p < 0.05) [28].
Broad-sense heritability (H2 ) was estimated according to Dini et al. [18] for resistance
to brown rot in fruits. The calculation was based on the data obtained in all seasons
evaluated for W and NW fruits, and the estimated environmental variance (σ̂2e ) divided by
three and two, respectively (environments number = seasons of evaluation). Narrow-sense
heritability (h2 ) was estimated from the regression of the average of parental phenotypic
values vs. offspring phenotypic values [29–31].
Genetic advance (GA%), also called response to selection or genetic progress was
estimated, and expressed in percentage of the population mean, using the formula:

Xs − Xo h2


GA% = ∗ 100 (1)
Xo

where: Xs mean of the selected genotypes; Xo original mean (base population); Xs − Xo = SD,
selection differential; h2 , narrow-sense heritability [29]. ‘Bolinha’ was then used as a
reference for the selection of the best genotypes.
 varying between 92–100% in the parents and between 83–99% in the progeny (Figure 1A).
However, 22 genotypes showed a BRI of less than 80%, being most of them on progeny
2012.52 (4 seedlings), 2012.66 (5 seedlings), and 2012.68 (5 seedlings).
When inoculation was made on NW fruits, there was a wide variability, between 18–
100% among parent plants and between 14–69% in the progeny (Figure 1B). The progeny
Agronomy 2022, 12, 2306 4 of 13
with the lowest means were 2012.52 (20%), 2012.66 (14%), 2012.68 (34%) and 2012.88 (30%).
A total of 43 seedlings presented less than 20% BRI, and most of them are part of the
aforementioned progeny (5, 5, 6, and 4 seedlings, respectively). It should be noted that the
3. Results2012.52 and 2012.66 are a product of a controlled cross between Conserva 947
progeny
Evaluation
and Conserva of wounded
1600, and in turn,fruits showed2012.68
the progeny that alland
genotypes were
2012.88 are susceptible
a product tocross
of the BRI,
varying between
between Conserva92–100%
1662 andin‘Maciel’,
the parents and between
parents 83–99%together
who presented, in the progeny (Figure 672,
with Conserva 1A).
However,
the lowest 22 genotypes
means. showed
In addition, theaprogeny
BRI of less than 80%,
2012.114 stoodbeing most
out with sixofseedlings
them on with
progeny
less
2012.52 (4 seedlings), 2012.66 (5 seedlings), and 2012.68 (5 seedlings).
than 20% of BRI, and all seedlings produced fruits with a marked pilosity.

Wounded Non-wounded
A
100
80
BRI (%)

60
40

20
0 ' C 577
Ca 'Ce 26

Co im 55

va 0
Ca 'M 62

at 59
'C a 1 '

Co erv ita'

ta '

t '

b '

' B 163

'
Co ser 672
Co erv 947

'S 506

ec 2
ec 0
'B 'M 540
Ca ata iel'

2D el'

ha
at to

ec o

ec st

Ru ena
er 60

N a 53
N a 48
N orã

N mi

TX im
sc rri

16
5

h 0

sc 13

in
sc ac
ns arr

a1
ns a 1
a1

r
ta
n a
ns va

un

RS o

ol
h

t
v
er
ns
Co

Parents
B
100

80
BRI (%)

60
40

20
0
59

20 8

20 6

20 3

20 9

20 1

20 2

20 6

20 8

20 8

20 1

20 6
2 0 .99

2 0 07

2 0 11
14
.2

.4

.6

.5

.6

.8

.3

.4
.3

.4

.6
.1

.1

.1

.1
09

12

12

12

12

12

12

12

12

12
12

12
08

12

12

12
20
20

Progenies
Figure 1. Brown
Figure 1. Brown rot incidence (BRI)
rot incidence (BRI) on
on wounded
wounded and
and non-wounded
non-wounded (W(W and
and NW)
NW) fruits
fruits of
of parents
parents
(A)
(A) and
andprogeny
progeny(B)
(B)average forfor
average three andand
three twotwo
seasons (2015–2016;
seasons 2016–2017;
(2015–2016; 2017–2018
2016–2017; and 2016–
2017–2018 and
2017; 2017–2018, respectively). ‘Bolinha’ was included as control. Embrapa Peach Breeding Pro-
2016–2017; 2017–2018, respectively). ‘Bolinha’ was included as control. Embrapa Peach Breeding
gram, Pelotas, Rio Grande do Sul, Brazil.
Program, Pelotas, Rio Grande do Sul, Brazil.

When inoculation was made on NW fruits, there was a wide variability, between
18–100% among parent plants and between 14–69% in the progeny (Figure 1B). The progeny
with the lowest means were 2012.52 (20%), 2012.66 (14%), 2012.68 (34%) and 2012.88 (30%).
A total of 43 seedlings presented less than 20% BRI, and most of them are part of the
aforementioned progeny (5, 5, 6, and 4 seedlings, respectively). It should be noted that
the progeny 2012.52 and 2012.66 are a product of a controlled cross between Conserva 947
and Conserva 1600, and in turn, the progeny 2012.68 and 2012.88 are a product of the cross
between Conserva 1662 and ‘Maciel’, parents who presented, together with Conserva 672,
the lowest means. In addition, the progeny 2012.114 stood out with six seedlings with less
than 20% of BRI, and all seedlings produced fruits with a marked pilosity.
Under the W treatment the seedlings the LA followed approximately normal distri-
bution. On the other hand, NW treatment exhibited seedling distribution like Lognormal
Agronomy 2022, 12, x FOR PEER REVIEW 5 of 13

Agronomy 2022, 12, 2306 5 of 13

Under the W treatment the seedlings the LA followed approximately normal distri-
bution. On the other hand, NW treatment exhibited seedling distribution like Lognormal
or Weibull, concentrated in classes of lower LA (0–10 and 10–20% of the area affected by
or Weibull, concentrated in classes of lower LA (0–10 and 10–20% of the area affected by
lesion) (Figure 2).
lesion) (Figure 2).

1.0 2008.159 W A 2009.38 W B 2012.26 W C 2012.43 W D

2008.159 NW 2009.38 NW 2012.26 NW 2012.43 NW
Relative frequency

0.8 PF PM P F PM PF P M PF P M
0.6 W 41% 27% W 27% 41% W 30% 44% W 44% 30%
NW 11% 13% NW 13% 11% NW 21% 25% NW 25% 21%
0.4

0.2

0.0
0 10 20 30 40 50 60 70 80 90100 0 10 20 30 40 50 60 70 80 90100 0 10 20 30 40 50 60 70 80 90100 0 10 20 30 40 50 60 70 80 90100

1.0 2012.49 W E 2012.61 W F 2012.52 W G 2012.66 W H

2012.49 NW 2012.61 NW 2012.52 NW 2012.66 NW
Relative frequency

0.8 PF P M PF P M PF PM PF P M
0.6 W 19% 41% W 41% 19% W 12% 19% W 19% 12%
NW 7% 11% NW 11% 7% NW 4% 3% NW 3% 4%
0.4

0.2

0.0
0 10 20 30 40 50 60 70 80 90100 0 10 20 30 40 50 60 70 80 90100 0 10 20 30 40 50 60 70 80 90100 0 10 20 30 40 50 60 70 80 90100

1.0 2012.68 W I 2012.88 W J 2012.31 W K 2012.46 W L

2012.68 NW 2012.88 NW 2012.31 NW 2012.46 NW
Relative frequency

0.8 P F PM P F PM P F PM P F PM
0.6 W 13% 22% W 22% 13% W 37% 44% W 74% 22%
NW 12% 7% NW 7% 12% NW 31% 58% NW 56% 7%
0.4

0.2

0.0
0 10 20 30 40 50 60 70 80 90100 0 10 20 30 40 50 60 70 80 90100 0 10 20 30 40 50 60 70 80 90100 0 10 20 30 40 50 60 70 80 90100

1.0 2012.99 W M 2012.107 W N 2012.111 W O 2012.114 W P

2012.99 NW 2012.107 NW 2012.111 NW 2012.114 NW
Relative frequency

0.8 PF P M PF PM PF P M P F PM
0.6 W 60% 59% W 75% 31% W 43% 26% W 34% 27%
NW 13% 53% NW 49% 19% NW 44% 13% NW 24% 7%
0.4

0.2

0.0
0 10 20 30 40 50 60 70 80 90100 0 10 20 30 40 50 60 70 80 90100 0 10 20 30 40 50 60 70 80 90100 0 10 20 30 40 50 60 70 80 90100
LA (%) LA (%) LA (%) LA (%)

Figure 2. Relative frequency histograms of progeny of 16 families (A–P) classified by lesion area
Figure 2. Relative frequency histograms of progeny of 16 families (A–P) classified by lesion area
(LA) expressed as percentage of the fruit that was affected by the brown rot lesion, on wounded (W)
(LA) expressed as percentage of the fruit that was affected by the brown rot lesion, on wounded
and non-wounded (NW) fruits. The mean values of female (Pf) and male (Pm) parents for each pop-
(W) and are
ulation non-wounded (NW) fruits.
indicated. Embrapa The Breeding
Peach mean values of female
Program, (Pf ) and male
2015–2016, (Pm ) parents
2016–2017 for each
and 2017–2018
population are indicated. Embrapa Peach Breeding Program, 2015–2016, 2016–2017 and 2017–2018
growing seasons, Pelotas, Rio Grande do Sul, Brazil. Progeny: 2008.159 (A), 2009.38 (B), 2012.26 (C),
growing seasons,
2012.43 (D), Pelotas,
2012.49 Rio Grande
(E), 2012.61 do Sul, Brazil.
(F), 2012.52 Progeny:
(G), 2012.66 (H),2008.159
2012.68 (A), 2009.38 (J),
(I), 2012.88 (B), 2012.31
2012.26 (K),
(C),
2012.43 (D),2012.99
2012.46 (L), 2012.49(M),
(E), 2012.107
2012.61 (F),
(N),2012.52 (G),
2012.111 (O)2012.66 (H), 2012.68
and 2012.114 (P). (I), 2012.88 (J), 2012.31 (K),
2012.46 (L), 2012.99 (M), 2012.107 (N), 2012.111 (O) and 2012.114 (P).
The 2012.52 and 2012.66 progeny (Figure 2G,H), grouped the highest percentage of
The 2012.52
seedlings andtwo
in the first 2012.66 progeny
categories (Figure
of LA 2G,H),
(58 and 35%,grouped the highest
respectively) when thepercentage of
fruits were
seedlings in the NW
W, and under first two categories
treatment of LA (58 and
all genotypes 35%, respectively)
of these when the
progeny, together fruits
with the were W,
2012.61
and under(Figure
progeny NW treatment allgrouped
2F), were genotypes of these
within theprogeny,
first twotogether with(0–10
categories the 2012.61 progeny
and 10–20% of
(Figure 2F), were grouped within the first two categories (0–10 and 10–20%
the area affected by lesion). The parents of 2012.52 and 2012.66 progeny (Conserva 947 of the area
affected by lesion).
and Conserva 1600)Thehadparents of 2012.52
the lowest meansand 2012.66
of LA progeny
for both (Conserva
W fruits (12 and947
19% and Con-
respec-
serva 1600) had the lowest means of LA for both W fruits (12 and 19% respectively)
tively) and NW treatment (4 and 3%, respectively). Conserva 672 selection, one of the par- and
NW treatment (4 and 3%, respectively). Conserva 672 selection, one of
ents of 2012.61 progeny, also presented low LA value (19 and 7%, for W and NW fruits, the parents of
2012.61 progeny, also presented low LA value (19 and 7%, for W and NW fruits, respec-
Agronomy 2022, 12,
Agronomy 2022, 12, 2306
x FOR PEER REVIEW 6 6of
of 13
13

respectively),
tively), as wellasaswell as the parents
the parents of 2012.68
of 2012.68 and 2012.88
and 2012.88 progenyprogeny
(Conserva (Conserva
1662 and1662 and
‘Maciel’)
‘Maciel’) of LA
of LA 13 and 22%13 (W)
and and
22% 12(W)andand7%12(NW),
and 7% (NW), respectively
respectively (Figure 2I,J).
(Figure 2I,J).
The 2012.26
2012.26andand2012.43
2012.43 progeny
progeny (Figure
(Figure 2C,D)2C,D)
and the and the 2012.99,
2012.99, 2012.1072012.107
and 2012.111and
2012.111
nectarinenectarine progeny
progeny (Figure (Figurerespectively)
2L,M,N, 2L, Figure 2M, wereandtheFigure 2N, respectively)
most susceptible to brown were
rottheon
most susceptible
W fruits, to brown
with a high rot on Woffruits,
concentration with in
seedlings a high concentration
the categories of 40of
toseedlings
70% of the inarea
the
categories
affected byofthe40lesion.
to 70% of the area affected by the lesion.
relation to
In relation toSPP,
SPP,high
highvariability
variabilitywas wasdetected
detected forfor both
both WW and and
NW NW fruits,
fruits, withwithin-
intervals
tervals forfor the
the parentsbetween
parents between16–96%
16–96%andand0–94%,
0–94%,respectively
respectively(Figure
(Figure 3A),
3A), and
and aa range
of 14–69% and and 0–35%
0–35% for
for WW and
and NWNW fruits
fruits of
of the
the progeny, respectively (Figure 3B). The
2012.66 progeny
2012.52 and 2012.66 progeny had had only
only 20
20 and
and 14%
14% ofofSPP,
SPP, respectively,
respectively, on W fruits, while
no seedlings
seedlingswithwithSPP
SPPononNW NWfruits werewere
fruits observed in both
observed in progenies. Secondly,
both progenies. the 2012.68
Secondly, the
and 2012.88 progeny had less than 35% of SPP on W fruits and, the 2012.61
2012.68 and 2012.88 progeny had less than 35% of SPP on W fruits and, the 2012.61 prog- progeny, stood
out with
eny, stoodlessoutthan
with11%
lessofthan
SPP11%on NW fruits.
of SPP Among
on NW theAmong
fruits. parents,thethose with those
parents, lowerwithSPP
mean SPP
lower for W andfor
mean NWWfruits
and NW were Conserva
fruits 947 and Conserva
were Conserva 1600 (parents
947 and Conserva of 2012.52
1600 (parents of
and 2012.66),
2012.52 ConservaConserva
and 2012.66), 672 (parent 672of(parent
2012.49ofand 2012.61),
2012.49 and and Conserva
2012.61), 1662 (parent
and Conserva 1662of
2012.68 of
(parent and 2012.88)
2012.68 and(Figure
2012.88)3A).
(Figure 3A).

Wounded Non-wounded A
100

80
SPP (%)

60

40

20

0
' ' ' ' ' ' ' '
526 rito 055 rita 672 947 600 662 ciel 359 577 rão 506 ist 532 480 540 ena mel 163 nha
r r o m r i i
a 1 e a 1 a va va a 1 a 1 a a 1 a 1 h ta n ta ta ta o b 2D ol
erv 'C scat him ser ser erv erv 'M scat scat 'C Nec 'Su Nec Nec Nec 'M Ru TX 'B
ns C n n s s
Ca ' Co Co Con Con Ca Ca RS
Co Parents
'B
100 B

80
SPP (%)

60

40

20

0
59 38 26 43 49 61 52 66 68 88 31 46 99 07 11 14
8.1 09. 12. 12. 12. 12. 12. 12. 12. 12. 12. 12. 12. 2.1 2.1 2.1
20
0 20 20 20 20 20 20 20 20 20 20 20 20 201 201 201
Progenies
Figure
Figure 3.
3.Sporulation
Sporulationpresence (SPP)
presence on on
(SPP) wounded andand
wounded non-wounded fruitsfruits
non-wounded of parents (A) and
of parents (A)prog-
and
eny (B) averages for three and two seasons (2015–2016; 2016–2017; 2017–2018 and 2016–2017; 2017–
progeny (B) averages for three and two seasons (2015–2016; 2016–2017; 2017–2018 and 2016–2017;
2018, respectively). ‘Bolinha’ was included as control. Embrapa Peach Breeding Program, Pelotas,
2017–2018, respectively). ‘Bolinha’ was included as control. Embrapa Peach Breeding Program,
Rio Grande do Sul, Brazil.
Pelotas, Rio Grande do Sul, Brazil.
For SPA, on W and NW fruits, the seedlings followed approximately a Lognormal or
For SPA, on W and NW fruits, the seedlings followed approximately a Lognormal or
Weibull distribution,with
Weibull distribution, witha ahigh
highconcentration
concentration
in in
thethe lower
lower categories,
categories, mainly
mainly the the
firstfirst
one
one (0–10% of the area affected by sporulation) (Figure 4). The only progeny that
Agronomy 2022, 12,
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x FOR PEER REVIEW 7 7of
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13

(0–10% of the
presented an area affected by normal
approximately sporulation) (Figure were
distribution 4). Thethose
only of
progeny that presented
nectarines, an
on W fruits
approximately
(Figure 4M–O). normal distribution were those of nectarines, on W fruits (Figure 4M–O).

1.0 2008.159 W A 2009.38 W B 2012.26 W C 2012.43 W D

2008.159 NW 2009.38 NW 2012.26 NW 2012.43 NW
Relative frequency

0.8 PF PM PF P M P F PM PF PM
0.6 W 41% 27% W 27% 41% W 30% 44% W 44% 30%
NW 11% 13% NW 13% 11% NW 21% 25% NW 25% 21%
0.4

0.2

0.0
0 10 20 30 40 50 60 70 80 90100 0 10 20 30 40 50 60 70 80 90100 0 10 20 30 40 50 60 70 80 90100 0 10 20 30 40 50 60 70 80 90100

1.0 2012.49 W E 2012.61 W F 2012.52 W G 2012.66 W H

2012.49 NW 2012.61 NW 2012.52 NW 2012.66 NW
Relative frequency

0.8 PF P M PF P M P F PM PF PM
0.6 W 19% 41% W 41% 19% W 12% 19% W 19% 12%
NW 7% 11% NW 11% 7% NW 4% 3% NW 3% 4%
0.4

0.2

0.0
0 10 20 30 40 50 60 70 80 90100 0 10 20 30 40 50 60 70 80 90100 0 10 20 30 40 50 60 70 80 90100 0 10 20 30 40 50 60 70 80 90100

1.0 2012.68 W I 2012.88 W J 2012.31 W K 2012.46 W L

2012.68 NW 2012.88 NW 2012.31 NW 2012.46 NW
Relative frequency

0.8 PF P M PF P M PF PM P F PM
0.6 W 13% 22% W 22% 13% W 37% 44% W 74% 22%
NW 12% 7% NW 7% 12% NW 31% 58% NW 56% 7%
0.4

0.2

0.0
0 10 20 30 40 50 60 70 80 90100 0 10 20 30 40 50 60 70 80 90100 0 10 20 30 40 50 60 70 80 90100 0 10 20 30 40 50 60 70 80 90100

1.0 2012.99 W M 2012.107 W N 2012.111 W O 2012.114 W P

2012.99 NW 2012.107 NW 2012.111 NW 2012.114 NW
Relative frequency

0.8 PF P M PF PM PF PM PF PM
0.6 W 60% 59% W 75% 31% W 43% 26% W 34% 27%
NW 13% 53% NW 49% 19% NW 44% 13% NW 24% 7%
0.4

0.2

0.0
0 10 20 30 40 50 60 70 80 90100 0 10 20 30 40 50 60 70 80 90100 0 10 20 30 40 50 60 70 80 90100 0 10 20 30 40 50 60 70 80 90100
SPA (%) SPA (%) SPA (%) SPA (%)

Figure 4. Relative frequency histograms of progeny of 16 families (A–P) classified by sporulation

Figure 4. Relative frequency histograms of progeny of 16 families (A–P) classified by sporulation
area (SPA) expressed as percentage of the fruit that was affected by the brown rot sporulation, on
area (SPA) expressed as percentage of the fruit that was affected by the brown rot sporulation, on
wounded (W) and non-wounded (NW) fruits. The mean values of female (Pf) and male (Pm) parents
wounded (W) and non-wounded
for each population (NW)
are indicated. fruits. Peach
Embrapa The mean valuesProgram,
Breeding of female 2015–2016,
(Pf ) and male (Pm ) parents
2016–2017 and
for each population
2017–2018 are indicated.
growing seasons, Pelotas,Embrapa
Rio Grande Peach
do Breeding Program,
Sul, Brazil. Progeny:2015–2016, 2016–2017
2008.159 (A), 2009.38 and
(B),
2017–2018
2012.26 (C),growing
2012.43 seasons, Pelotas,
(D), 2012.49 (E), Rio Grande
2012.61 (F), do Sul, Brazil.
2012.52 Progeny:
(G), 2012.66 (H),2008.159
2012.68(A),
(I), 2009.38
2012.88 (B),
(J),
2012.26 (K),
2012.31 (C), 2012.46
2012.43(L),
(D),2012.99
2012.49(M),
(E),2012.107
2012.61 (N),
(F), 2012.52
2012.111(G),
(O) 2012.66 (H), 2012.68
and 2012.114 (P). (I), 2012.88 (J),
2012.31 (K), 2012.46 (L), 2012.99 (M), 2012.107 (N), 2012.111 (O) and 2012.114 (P).
The maternal effect was analyzed (the contrast between the F1 progeny versus its
The maternal
reciprocal) effect
for all the was analyzed
parameters (theLA,
(BRI, LD, contrast between
SPP, SPD, and the F1 and
SPA), progeny
in allversus its
cases the
reciprocal) for all the parameters (BRI, LD, LA, SPP, SPD, and SPA), and in all
differences were not significant (p > 0.05) indicating that there is no maternal effect in- cases the
differences
volved were
in the not significant
transmission (p >trait.
of this 0.05) indicating that there is no maternal effect involved
in theHtransmission of this trait.
2 estimates were from medium to low, for the different parameters, ranging be-

tween H230.1–51.7%
estimates were
on Wfrom medium
fruits to low, for
and between the different
25.0–40.5% on parameters, ranging
NW fruits. The between
h2 estimates
30.1–51.7% on W fruits 2
varied depending on theand
year between 25.0–40.5%
of evaluation, on obtained
but the NW fruits. The were
means h estimates
betweenvaried
18.3–
depending on the year of evaluation, but the obtained means
41.6% for W fruits and between 18.7–33.9% for NW fruits (Table 2). were between 18.3–41.6% for
W fruits and between 18.7–33.9% for NW fruits (Table 2).
Agronomy 2022, 12, 2306 8 of 13

Table 2. Broad-sense (H2 ) and narrow-sense (h2 ) estimated heritability for incidence and severity of
M. fructicola on wounded and non-wounded peach fruits, Embrapa Peach Breeding Program, Pelotas,
Rio Grande do Sul, Brazil.

h2 (%)
Fruits Traits * H2 (%)
2015–2016 2016–2017 2017–2018 Mean
BRI 42.5 35.8 37.6 31.1 34.8
LD 51.7 47.1 42.1 35.5 41.6
LA 44.7 33.5 41.1 25.5 33.4
Wounded
SPP 27.5 23.2 18.0 13.8 18.3
SPD 41.2 33.0 39.3 35.9 36.1
SPA 30.1 21.0 28.9 18.5 22.8
BRI 26.7 - 20.2 17.1 18.7
LD 40.5 - 34.6 30.1 32.4
Non- LA 29.8 - 28.6 20.2 24.4
wounded SPP 25.0 - 20.1 11.8 16.0
SPD 31.1 - 29.3 19.9 24.6
SPA 23.6 - 21.8 16.9 19.4
* BRI, brown rot incidence; LD, lesion diameter; LA, lesion area; SPP, sporulation presence; SPD, sporulation
diameter; SPA, sporulation area.

Using the ‘Bolinha’ as the cut-off point for resistance selection (Figure S1), the genetic
advances obtained by the Embrapa Peach Breeding Program were estimated. The selection
differential (SD) is the subtraction of the selected genotypes mean (Xs ) minus the original
mean (Xo ).
The GA% associated with each variable was estimated for W and NW fruits, separately
(Table 3). The selected genotype number (SGN) was between 11 and 76, and GA% estimates
were between −5.2 to −30.2% on W fruits. On the other hand, the SGN on NW fruits was
between 53 and 93, and GA% estimates were between −15.0 and −27.3%.

Table 3. Genetic advancement by selection of genotypes better than or equal to ‘Bolinha’.

Wounded Non-Wounded
BRI LD LA SPP SPD SPA BRI LD LA SPP * SPD * SPA *
Xo 96.3 29.5 39.7 60.6 14.6 15.9 54.0 12.8 16.6 19.4 4.5 5.4
‘Bolinha’ 94.3 20.2 13.3 37.1 5.5 2.1 30.0 6.5 4.0 0 0 0
Xs 81.8 14.5 8.1 15.8 2.4 0.5 11.0 1.9 0.9 0 0 0
SD −14.5 −15.0 −31.6 −44.8 −12.2 −15.5 −43.0 −10.9 −15.7 −19.4 −4.5 −5.4
SGN 58 21 11 76 63 41 53 59 56 93 93 93
h2 0.35 0.42 0.33 0.18 0.36 0.23 0.19 0.32 0.24 0.16 0.25 0.19
GA (direct) −5.0 −6.2 −10.5 −8.2 −4.4 −3.5 −8.1 −3.5 −3.8 −3.2 −1.1 −1.0
GA% −5.2 −21.1 −26.5 −13.5 −30.2 −22.1 −15.0 −27.3 −22.8 −16.4 −25.0 −18.6
BRI, brown rot incidence; SPP, sporulation presence; LD, lesion diameter; SPD, sporulation diameter; LA, lesion
area; SPA, sporulation area; h2 , mean of narrow-sense heritability; SD, selection differential; SGN, selected
genotypes number; Xs , mean of the selected genotypes; Xo , original mean (base population); GA (direct), direct
response; GA%, genetic advance (selection response in percentage of the population mean); * ’Bolinha’ value = 0.

Using the cultivar Bolinha as control, four seedlings (2012.52.17, 2012.52.2, 2012.66.11,
and 2012.68.24) and a selection (Conserva 947) were identified, with better results in all
variables of incidence and severity to brown rot (BRI, LD, LA, SPP, SPD, and SPA) on W
fruits. In the case of NW fruits, 40 seedlings and three selections (Conserva 947, Conserva
1600, and Conserva 672) had equal or better results than ‘Bolinha’.

4. Discussion
One of the top priorities of the Embrapa Peach Breeding Program is disease resistance,
which is justified by the weather conditions of the area that is one the most important
in Brazil, for stone fruit production. The climate favors also the incidence of insect pests
Agronomy 2022, 12, 2306 9 of 13

causing damage to the fruit epidermis. Thus, the tests using W fruits were especially
important and proved to be suitable to identify resistance sources.
Exploring sources of brown rot resistance in an interspecific population of
almond × peach, Baró-Montel et al. [13] reported similar results to those of the present
study regarding BRI, where close to 100% of W fruits developed the disease, and NW
fruits varied between 0–80% of BRI. On the other hand, working with NW fruits, BRI
averages were reported between 60–100% [26] and between 50–100% [27]. These three
studies performed the evaluation at 120 HAI and in the present study evaluations were
made at 72 HAI.
In inoculation without wounding, the lower development of the disease may be due to
the delay in fungus infection and/or in the lower probability of conidia having a successful
penetration of fruit skin. Although visually the fruits appeared to be undamaged, there
may be microcracks where the fungus could infect. In the case of M. fructicola, it has been
reported that the wound is necessary and is the main gateway for infection [25,32–34]. Thus,
the conidia number that would have success in the infection, in the case of inoculation
with a wound would be much greater in relation to inoculation without a wound. For
this reason, the standard error bars are much smaller in the evaluations with injury when
compared to the uninjured fruit evaluations, both for BRI in the parents and in the progeny
(Figure 1). This indicates that the sample means were more reliable when the wound was
used, and this methodology is recommended, since microcracks of diverse origin can exist
and are highly associated with the environmental conditions and not with the genotype,
presenting a disparity between the evaluated seasons [13,27,35,36].
The 2012.114 progeny, with 46% BRI on NW fruits, had six seedlings with less than
20% of BRI (Figure 1B). This progeny was characterized by producing fruits with a high
pilosity, and this characteristic associated with unwounded inoculation can be considered a
structural barrier, hindering the development and infection of M. fructicola conidia in the
fruit [22,37]. However, in stone fruits, the subject is controversial, since there are reports
where the trichomes favor the infection of Monilinia spp. [8,38,39]. These studies mention
that microcracks at the base of the trichomes may result in entry points of the fungus. In
addition, the high density and length of the trichomes in some genotypes of the progeny
2012.114 can have caused a failure in the technique of inoculation, since the drop with a
conidial suspension of M. fructicola may have dried before coming into contact with the
fruit skin.
In the 2012.107 and 2012.111 nectarine progeny, erratic behavior was observed when
the fruits were evaluated without wounds (Figure 2N,L). Even though a large number of
fruits had no BRI, it was not associated with a specific genotype. This can be explained by
the presence of waxes associated with the nectarine’s cuticle, which may be considered
a structural barrier to fungal infection [33,35,37]. However, it can also be an error in the
inoculation technique, because a more waxy surface can cause the drop containing the
conidial suspension of M. fructicola to simply drain from the fruit or dry without having
sufficient time of contact with the fruit skin, so fungus did not infect.
High variability related to sporulation variables such as SPP (Figure 3) and SPA
(Figure 4), was detected both for W and NW fruits. In a study in which NW fruits were
inoculated with M. laxa, and evaluated 120 HAI, Obi et al. [27] obtained averages were
higher than in the present study (above 80%) of fruits with fungus sporulation (colonized),
probably due to the longer incubation time and/or genetic differences. Genotypes that had
rotted, but did not result in fungus sporulation, are of great epidemiological for the disease,
reducing the secondary inoculum in orchard [5,22]. Among the more than 300 genotypes
evaluated in this study, 23 (W) and 93 (NW) did not exhibit sporulation in any of their
fruits, evaluated for three and two years, on W and NW fruits, respectively.
No maternal significant effect was detected in the inheritance of the characters associ-
ated with brown rot resistance. The use of reciprocal crosses is the simplest evidence of
testing the maternal effect, since they would produce genetically similar but phenotypically
different individuals [23,31], if there was indeed a significant maternal effect. In stone fruits,
Agronomy 2022, 12, 2306 10 of 13

there are rare cases where a possible maternal effect has been mentioned [40,41] or maternal
inheritance [42–44] and has never been associated with resistance to pests or diseases.
All H2 and h2 estimates were from medium to low and, considering that the peach
resistance to brown rot is quantitative [13,19,20], the parents selection based on their
phenotype may be considered reasonable to not very effective. Studying the reaction to
M. fructicola on wounded peach fruits from the Embrapa Peach Breeding Program’s work
collection, Scariotto [17] estimated the H2 of LD and SPD at 50 and 13%, respectively.
The first estimate was similar to the present study (51.7%), but the second estimate was
considerably lower (41.2%). On the other hand, testing the response on unwounded
peach fruits of Embrapa Peach Breeding Program populations [45] estimated the BRI H2 at
64%, which is different from this study where for the same variable and under the same
conditions it was 26.7%. Although the site where these experiments were done was the
same, there were differences in methods, climatic conditions, and genotypes. Considering
these heritability estimates (low to medium values), moderate genetic advance with great
environmental influence can be expected due to selection for the characters associated with
brown rot resistance [29].
The lowest GA% was associated with BRI on W fruits (−5.2%), due to the low vari-
ability associated with this character in the studied populations. GA% of −13.5 to −30.2%,
were estimated for the other variables on W fruits. On the other hand, for NW fruits, GA%
estimates were between −15.0 to −27.3%, and in the case of the selected genotypes with
respect to sporulation, there were those that presented 0 (SPP, SPD, and SPA) as ‘Bolinha’.
There were not find references, in the literature, regarding genetic advances (genetic
gains or response to selection) in relation to disease resistance and/or pests in Prunus spp.
Estimating the heritability and predicted selection response of quantitative traits in peach,
Souza et al. [46] obtained predictions from 4.61–61.25% on fruit quality characteristics
(fruit mass, soluble solids, acidity, fruit length, and others) and between 20.44–95.00%
for phenological characters (full bloom, ripening and fruit development period). In the
same way, Chandrababu and Sharma [47] studying the heritability and genetic gain in
several characteristics of almond (Prunus dulcis), obtained values of genetic gain between
37.98–187.27% for growth and yield characters, 33.77–132.79% for flowering and fruiting
characters, and 23.08–74.36% for fruit and kernel characters. Polygenic traits have low
heritability and high environmental influence, and the genetic gain expected by selection
based on the phenotype is generally moderate to low [29,48].
Among the five genotypes with the best results on W fruits, there were three seedlings
and one selection related directly to ‘Bolinha’. The advanced selection Conserva 947
was selected from a cross between ‘Bolinha’ × P60-22 (Mexican polen) and, the seedlings
(2012.52.17, 2012.52.2, and 2012.66.11) originated from the cross between
Conserva 947 × Conserva 1600 (2012.52), and its reciprocal (2012.66). Among the 43 se-
lected genotypes with the best results on NW fruits, there were 11 genotypes related to
‘Bolinha’, five seedlings of 2012.52 progeny and five of 2012.66 progeny, in addition to
selection Conserva 947. Besides these genotypes, the other progeny that participated with
a higher number of selected genotypes were 2012.68 (one and six, on W and NW fruits, re-
spectively) and 2012.88 (four, on NW fruits), progeny originated from the crossing between
Conserva 1662 × ‘Maciel’ and their reciprocal cross, respectively. Another of the selected
genotypes, Conserva 672 advanced selection, had previously been reported as being one of
the genotypes with the best results on non-wounded fruits [45].
All selected genotypes with and without a wound, had equal or better results than
‘Bolinha’ for brown rot resistance in fruits and, several of them, mainly the selections
(Conserva 947, Conserva 1600, and Conserva 672) have superior fruit quality to ‘Bolinha’,
demonstrating the progress of the Embrapa Peach Breeding Program, not only regarding
resistance to M. fructicola.
Agronomy 2022, 12, 2306 11 of 13

5. Conclusions
The studied populations present variability regarding brown rot resistance, without
any evidence of maternal effect. The heritability of brown rot resistance in fruits is medium
to low. Selection of genotypes based on phenotypes better or equal to ‘Bolinha’, allowed
estimate of moderate to low genetic advances for brown rot resistance. The Embrapa Peach
Breeding Program is achieving genetic advances in fruit resistance to M. fructicola, currently
having several genotypes comparable to ‘Bolinha’ but with better fruit quality.

Supplementary Materials: The following supporting information can be downloaded at: https:
//www.mdpi.com/article/10.3390/agronomy12102306/s1. Figure S1: Hypothetical distribution of
all evaluated genotypes from the 16 populations; selection was based on the incidence and severity
means of ‘Bolinha’. The selection differential (SD) is the subtraction of the selected genotype’s mean
(Xs ) minus the original mean (Xo ).
Author Contributions: Conceptualization, M.d.C.B.R. and B.U.; methodology, B.U., S.S. and M.D.;
software, M.D.; validation, M.d.C.B.R. and B.U.; formal analysis, M.D.; investigation, M.D. and
S.S.; resources, M.d.C.B.R.; data curation, M.D.; writing—original draft preparation, M.D.; writing—
review and editing, M.D.; visualization, M.D. and M.d.C.B.R.; supervision, M.d.C.B.R. and B.U.;
project administration, M.d.C.B.R.; funding acquisition, M.D. All authors have read and agreed to the
published version of the manuscript.
Funding: This research was funded by National Agency for Research and Innovation (ANII-Uruguay)
awarding the master’s degree scholarship and the Coordination for the Improvement of Higher
Education Personnel (CAPES-Brazil) awarding the first author’s doctoral scholarship.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Acknowledgments: Authors thank Embrapa staff, especially Everton Pederzolli, Maicon Bönemann,
and Gilberto Kuhn, and to the colleagues and friends who at some point helped with the practical
work: Priscila Monalisa Marchi, Silvia Carpenedo, Carol Silveira, Julia Ritter, Diego Borges, and
Leonardo Milech.
Conflicts of Interest: The authors declare no conflict of interest.

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