Introduction
Citrus fruit production has seen notable growth in recent
years. According to the recent statistics reported by USDA (2021), tangerines and their hybrids constitute the most
important and predominant group with a production of 1.2 Million tons (Mt) and
exports exceeded 500000 tons. In 2019, Morocco holds the third place on
tangerines and mandarins exportation (USDA 2019).
Despite its importance, the orchards suffer from a range of fungal diseases,
especially, Alternaria brown spot (ABS) caused by the tangerine pathotype, Alternaria
alternata (Fr.) Keissl.
(Stewart et al. 2014; Azevedo et al.
2019). Recently, there was report of occasional association of A. arborescens pathogen with Alternaria brown spot on
citrus (Moosa et al. 2020; Wang et al.
2021; Zelmat et al. 2021). The ABS is widely distributed
throughout the world (Elena 2006) and is
considered distinct from other Alternaria diseases on citrus including
Alternaria black rot (ABR) and Alternaria leaf spot of rough lemon. This
disease causes necrotic lesions on leaves, twigs and fruits in the pre-harvest
stage (Aiello et al. 2020).
Moreover, the ABS can be expressed after fruit harvesting and provokes
different symptoms as stem-end rot infection and leads to serious value loss of
the mandarins (Garganese et al. 2016; Saito
and Xiao 2017). The produced necrotic areas are commonly associated with
the ACT host-specific toxin production which distorts profoundly the plasma
membrane permeability of the plant hosts cells (Tsuge
et al. 2013; Ma et al. 2019).
Until
now, the main strategy used to control the ABS over the world is based
essentially on the fungicide applications depending on the genotype and climate
(Vega et al. 2012; Kim et al.
2017). In Morocco, copper, copper oxychloride and copper hydroxide were
applied heavily to managed the Alternaria diseases on citrus (Lahlali et al. 2021). However, current
studies conducted on Florida and Brazilian orchards of tangerines and their
hybrids to assess the A. alternata sensitivity
to chemical compounds demonstrate that a large number of A. alternata isolates from citrus were resistant to the
fungicides based on copper and quinone outside inhibitor (QoI)
(Chitolina et al. 2021). In
addition to the emergence of resistant A. alternata
strains, intensive applications of fungicides have harmful and hazardous consequences
for animals, humans and the environment (Vicent et
al. 2009). In fact, several studies have been carried out to find a
sustainable solution and develop a long-term strategy to combat this disease,
in particular, by researching new ABS-resistant cultivars (Campos et al. 2017; Turgutoğlu and Baktir
2019; Costa et al. 2020). The genotypes are classified mainly
according to their level of resistance. Dancy, Fortune and Murcott
were reported as susceptible to ABS, whereas Afourer
and Carvalhais were considered among the resistant
cultivars (Arlotta et al. 2020).
In fact, the tolerance of the resistant cultivars to ABS is controlled by a
single recessive locus (ABSr) (Gulsen et al. 2010; Cuenca et al. 2013). The present
study aimed to determine the susceptibility or resistance of 10 cultivated
varieties of mandarins to A. alternata fungus
isolated from citrus basing on the in vitro and in vivo
inoculation.
Materials and Methods
Procurement of A. alternata
Isolate
A Moroccan single isolate of A. alternata
(MW616576) originating from symptomatic orange citrus fruit (Zelmat et al. 2021), was used in this
study (Fig. 1). The virulence of this fungal isolate was evaluated by
inoculating detached leaves of susceptible genotype, Citrus jambhiri ‘Rough lemon’. This examination was performed
in order to determine the capacity of the A. alternata
isolate to cause lesions and develop necrosis around the inoculated point
(Fig. 1).
Plant Materials
The
resistance to ABS was assessed for 10 varieties of mandarins/hybrids belonging
to the INRA experimental orchards (Table 1). These plants were selected based
both on their potential commercial value and on the availability of mature
fruits during the period of inoculation.
Preparation of Inoculum
The A. alternata
inoculum was prepared from young cultures of five days as described by Costa et al. (2020) with some
modifications. The conidia were collected by adding 10 mL of sterilized
distilled water containing Tween 20 (0.02%, v/v) to each plate and scraping
gently using a sterile scalpel. Then, the obtained suspension was filtered
through two layers of sterile paper to eliminate the mycelium fragments and
adjusted to 105 conidia mL-1 using a hemocytometer
technique.
Field Inoculation
Field bioassays were carried out over previously
selected trees of mandarins. Three plants of each cultivar were used in this
experiment. A total of nine young leaves (5–7 cm) and nine mature fruits from
each tree were marked before and inoculated manually by spraying appositively 2
mL of the conidia suspension per leaf/fruit (Pacheco
et al. 2012). Controls were inoculated with the same volume of
sterile distilled water. Thus, the inoculated samples were covered with a
transparent polyethylene bag whose interior was previously sprayed with sterile
distilled water to serve as a humid chamber (Reis
et al. 2007; Souza et al. 2009). The assessments were
performed four to seven days after inoculation (Souza
et al. 2009).
Inoculation of Detached Leaves
In vitro inoculations were performed in two consecutive years
(2019 and 2020) for all the tested cultivars. Young leaves measured 5–7 cm and
2–3 cm in length were sampled from the plants and inoculated immediately after
their harvest. Briefly, nine leaves per variety were disinfected in 1% of
hypochlorite sodium and placed individually in Petri dishes with a humid filter
paper and inoculated using the same volume of suspensions mentioned above.
Control leaves were sprayed only with distilled water and the plates were kept
at the temperature of 27°C under a 12 h photoperiod. Disease assessments were
performed 24 h, 48 h and 72 h after inoculation (Reis
et al. 2007).
Assessment Method
The severity of symptoms on the fruits was referred using
a specific scale described by Pacheco et al.
(2012) as shown in Table 2. For leaves, the severity of symptoms was
determined using a specific diagrammatic scale developed by Martelli et al. (2016) to assess the ABS
leaf symptoms (Table 3). The results were recorded by calculating the area
under the disease progression curve (AUDPC) (Campos
et al. 2017):
AUDPC = ∑ [((y1+y2)/2*(t2-t1)]
Where y1 and y2 are two consecutive evaluations carried %20295-300_files/image001.png)
out at times t1 and t2 respectively.
Results
Field Evaluation of Alternaria Brown Spot
In this experiment, the resistance level of ten
varieties of mandarins to A. alternata was
evaluated for both fruits and leaves. Field results showed that all studied
varieties were affected and responded to this pathogen. Symptoms were
%20295-300_files/image003.png)
Table 1: List of the mandarin cultivars used in this study, from
INRA of Morocco
|
Accession |
Description |
System/References |
|
Murcott Honey |
C. reticulata × C. Sinensis |
|
|
Bergamota |
Citrus reticulata |
Swingle system |
|
Dancy |
Seedling of Moragne tangierine |
|
|
Carvalhal |
Citrus reticulata Blanco |
Tanaka system |
|
Ananas |
Citrus reticulata Blanco |
Tanaka system |
|
Satsuma wase |
Citrus unshiu |
Tanaka system |
|
Vohanisahy Ifranica |
Citrus reticulata |
Swingle system |
|
Lee |
C. paradisi × tangerine |
Tanaka system |
|
Temple |
C. reticulata × C. sinensis |
Tanaka system |
|
Nadorcott |
Murcott tangor × Mandalina mandarin |
typically characteristic of ABS disease as shown in the
Fig. 2. On fruit, small necrotic spots were dispersed randomly in their surface
while the leaf lesions included large area brown to black areas according to
the severity incidence. In fact, the AUDPC values given in Table 4 indicate
that lesions on fruits were more severe than on leaves. The minimum values of
AUDPC were recorded by Vohanisahy Ifranica,
Lée, Temple and Nadorcott
cultivars, with 127.75, 125.88, 122.8, 87.63 on fruits, and with 40.69, 46.06,
32.88, 38.63 on leaves, respectively. The higher values of AUDPC were observed
on fruits of Murcott Honey, Bergamota
and Dancy and on leaves of Carvhalal, Ananas and
Satsuma Wase.
In vitro Evaluation of Alternaria Brown Spot
The detached-leaf method was repeated in two successive
years depending on the leaves size. The ABS severity of each cultivar was
calculated as AUDPCs (Table 4). Compared to the field inoculations, lesions on
leaves inoculated under controlled conditions (in vitro) were slightly
severe. On the other hand, results showed that the young leaves measuring 3 to
4 cm in size were more susceptible to ABS than those measuring 5–7 cm. The
symptoms were generally typical and appeared as irregular areas with a dark
brown color (Fig. 3). Based on the in vitro results, Nadorcott
and Temple seem the most resistant cultivars to A. alternata,
whereas high values of AUDPC were observed for Murcott,
Dancy, Carvalhal and Ananas.
Discussion
ABS is one of the severest diseases of mandarins (C.
reticulata) and their hybrids leading to significant economic %20295-300_files/image005.png)
Fig. 2: Fruits showing the typical symptoms of ABS after 7 days
of field inoculation (a); Temple (b); Ananas (c); Carvhalal (d); Vohanisahy Ifranica
%20295-300_files/image007.png)
Fig. 3: Leaves showing the typical symptoms of ABS after 48 h of
in vitro inoculation (a); Carvhalal (b); Dancy
(c); Ananas (d); Satsuma wase
Table 4: Area under the disease progress curve (AUDPC) of ABS in
2019 and 2020
|
Cultivars |
AUDPC |
||||
|
In field |
In
laboratory |
||||
|
Fruits |
Leaves
(5–7 cm) |
Leaves
(5–7 cm) |
Leaves
(2–3 cm) |
||
|
Murcott Honey |
201.2 |
51.42 |
149.34 |
222.18 |
|
|
Bergamota |
182.66 |
57.57 |
75.27 |
158.64 |
|
|
Dancy |
175.26 |
56.34 |
78.99 |
264.78 |
|
|
Carvhalal |
157.39 |
74.04 |
74.68 |
295.05 |
|
|
Ananas |
154.27 |
70.32 |
76.52 |
200.06 |
|
|
Satsuma Wase |
149.89 |
70.33 |
88.87 |
254.9 |
|
|
Vohanisahy Ifranica |
127.75 |
40.69 |
86.39 |
296.88 |
|
|
Lée |
125.88 |
46.06 |
49.35 |
150.59 |
|
|
Temple |
122.8 |
32.88 |
33.93 |
68.5 |
|
|
Nadorcott |
87.63 |
38.63 |
41.33 |
140.09 |
|
losses. Discovering and screening for resistant
cultivars to this disease became recently a major objective for several
researchers. In the present study, the evaluation of ABS resistance was carried
out in the field and on detached leaves of ten mandarins varieties using a
virulent A. alternata isolate. Results showed
a different degree of susceptibility among the tested cultivars. The ABS
symptoms were clearly expressed on fruits and young leaves inoculated
artificially with A. alternata pathogen. The
produced lesions were observed as brown to black-colored areas dispersed in the
leaf surface. Similar symptoms were detected on leaves of resistant and
susceptible mandarin hybrids inoculated with A. alternata
spores (Campos et al. 2017; Arlotta et
al. 2020). In the literature, it has been widely reported that the
development of lesions is mainly due to the host-selective ACT-toxin production
by A. alternata tangerines pathotype (Akimitsu et al. 2014). On the other
hand, the detached leaves were found highly susceptible to the disease than
those inoculated in the trees. This is in agreement with several studies
reporting the fast development of severe symptoms under controlled conditions
compared with the in vivo experiments (Souza
et al. 2009; Turgutoğlu and Baktir 2019). Azevedo et al. (2010) noted that the physiological defense of leaves
against pathogens is decreased and lost gradually after their detachment. On
the other hand, the resistance level assessment of the studied cultivars based
on the AUDPC, showed the high susceptibility to A. alternata
by Murcott Honey, Bergamota,
Dancy, Carvhalal, Ananas, Satsuma Wase and Vohanisahy Ifranica. Lee, Temple
and Nadorcott were recorded low severity and to be
resistant to ABS on leaves and fruits. In accordance, Dancy and Murcott were reported in previous studies as highly
susceptible genotypes to this disease and used as controls (Cuenca et al. 2013; Campos et al. 2017).
A current study demonstrates genetically that Nadorcott
mandarin (syn. Afourer) was moderately resistant to A.
alternata fungus (Arlotta
et al. 2020). Likewise, Reis et
al. (2007) and Souza et al. (2009) describe the Temple as
slightly susceptible to this fungus. This variety was also found resistant to
ABS in previous data (Cuenca et al. 2016).
Conclusion
Among the ten mandarin cultivars evaluated in this
study, Temple, Nadorcott and Lée
were revealed a medium resistance to A. alternata
pathogen. Furthermore, our findings indicate the greater susceptibility of the
detached leaves inoculated in the laboratory to the ABS disease. Hence, new
mandarins varieties should be tested with different A. alternata
isolates show a high pathogenic ability to cause disease in the citrus crops.
Acknowledgments
This study was supported by the grant from KAFACI (Project
Number: KAH 20180108), Rural Development Administration of Korea.
Author Contributions
All authors have read and
agreed to the published version of the manuscript.
Conflicts of Interest
No potential conflict of
interest was reported by the authors.
Data Availability
Accession No MW616576 is
already available in NCBI
Ethics Approval
Not applicable
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