Evaluation of 20 Citrus Varieties Resistance to Pseudofabraea

citricarpa

Quan Chen

1,2

, Wenjing Zhang

1,2

, Jiequn Ren

1,2

, Pingwei Xiang

1,2

and Jinhui He

Chongqing Three Gorges Academy of Agricultural Sciences, Wanzhou, 400401, Chongqing, China

College of Biology and Food Engineering, Chongqing Three Gorges University, Wanzhou, 404020, Chongqing, China

Plant Protection and Fruit Tree Technology Popularization Station in Wanzhou District of Chongqing, Wanzhou, 404155,

Chongqing, China

Keywords: Citrus Target Spot, Pseudofabraea citricarpa, Resistance Evaluation.

Abstract: Citrus target spot is one of the most destructive diseases on leaves, shoots and fruits of some citrus in China.

In this study, to evaluate the resistance against the disease, the separated leaf inoculation method was used

to inoculate Pseudofabraea citricarpa pathogen on abatial of citrus leaves under 10℃. Resistance of 20

citrus varieties were evaluated. The results indicated that among the 20 citrus varieties, 18 varieties were

classified into highly resistant varieties according to the diameter spots. Orah 091 was moderate resistant

and CRIC32-01 was resistant to Ps. citricarpa. The study could be used for the further study of citrus

resistance genes to Ps. Citricarpa and has given suggestions for structure in citrus production areas.

1 INTRODUCTION

Citrus target spot is a newly emerging citrus disease

caused by Pseudofabraea citricarpa (Yang, Fang,

Yu, Bi, Zhou, 2019); (Chen, 2016). And a newly

emerging leaf-spotting disease of citrus reported in

the year of 2004 which was occurred in late winter

and early spring in Chenggu County, Shanxi

Province of China (Zhu, Wang, Huang, Zhang, Li,

2012). This fungal pathogen could infect both

Satsuma mandarin (Citrus unshiu) and kumquat

(Fortunella margarita) in orchards (Zhu, Wang,

Huang, Zhang, Li, 2012). This disease caused

considerable economic losses in Wanzhou,

Chongqing City, Yichang City, Hubei Province and

Jishou City, Hunan Province, China in 2021 (Xiao,

Zeng, Wang, Cheng, Li, 2020). These morbidity

tendency demonstrated a trend of accelerating

propagation from north to south of China (Zhan,

2021). These incidence trend was the same as our

previous prediction of suitable area and risk analysis

for citrus target spot (Xu, Chen, 2020). Citrus target

spot occurs during late winter and early spring and

causes severe leaf spotting, defoliation or even

fruit-dropping and tree- dead. Once the disease

becomes epidemic, existing technology including

chemical agents, some agricultural control and

physical control are difficult to control effectively

(Xiao, Zeng, Wang, Cheng, Li, 2020); (Zhu, 2012).

Breeding resistant varieties is the most fundamental

means of this disease control.

At present, the research on citrus target spot is

still in the initial stage, mainly focusing on the

identification of pathogen, identification of

pathogenic factors and prediction of suitable areas

(Yang, Fang, Yu, Bi, Zhou, 2019). The SCAR

molecular detection provides a fast and easy method

for early identification of this disease (Yang, Hu,

2018). And the research integrated transcriptomic

and secretomic approaches revealed critical

pathogenicity factors in Ps. citricarpa inciting citrus

target spot (Yang, Fang, Yu, Bi, Zhou, 2019).

According to the latest research, citrus target spot is

at a high risk level in China, and the high and

middle suitable areas are mainly concentrated in the

citrus dominant area in the upper and middle reaches

of Yangtze River (Xu, Chen, 2020). However, there

is not a perfect and viable standardized system for

the identification of resistance to citrus target spot,

which leads to the slow process of screening

resistant germplasm and breeding resistant varieties.

Therefore, we took the average diameter of the

speckle and the incidence as the evaluation standard

for resistance (Chen, Liu, 2009); (Hu, Liu, 2015);

(Liu, Hu, 2013); (Zhang, Ding, 2013); (Ling, Huang,

2011); (Li, Zheng, 2009) and inoculated different

Chen, Q., Zhang, W., Ren, J., Xiang, P. and He, J.

Evaluation of 20 Citrus Varieties Resistance to Pseudofabraea Citricarpa.

DOI: 10.5220/0011212900003443

In Proceedings of the 4th International Conference on Biomedical Engineering and Bioinformatics (ICBEB 2022), pages 397-401

ISBN: 978-989-758-595-1

397

citrus varieties for identifying resistance to this

disease.

2 METHODS AND MATERIALS

Fungal Pathogens. Ps. citricarpa strains

Pc-WZBY1was isolated from Eureka lemon leaves

in Wanzhou District, Chongqing City. Direct tissue

isolation of causal agents was performed reported by

Zhu et al. (Zhu, Wang, Huang, Zhang, Li, 2012) The

materials were surface-sterilized with 75% ethanol

for 30 s, 1% NaOCl for 1 min, and then rinsed with

sterile distilled water for 5 times. Small sections (3 ×

5 mm) from the margins of diseased and healthy

tissues were placed onto potato dextrose agar (PDA)

in petri dishes. Small drops of suspension were

placed on a glass slide and examined under a

dissecting microscope at ×20 magnification. Drops

that contained only a single spore were transferred to

fresh PDA plates. The plates were incubated at 20°C

until the mycelium covered approximately

three-quarters of the plates (about 15 to 20 days).

Pure single-spore cultures grew on PDA were then

transferred onto fresh PDA and stored at 4°C for

further study.

Citrus Varieties. C.grandis (Shatianyou,

Hongbaoshiyou, Taiguoqingyou, Dianjiangwanyou,

DianjiangBaiyou, Liangpingyou, Sanhongyou),

C.sinensis (Tarocco Blood Orange NO.8, Tarocco

Blood Orange NO.9, Newhall Navel Orange),

C.reticulata (Shiranui, Aiyuan NO.38, Daya

mandarin, Tango, Orah 091, Gold Nugget) and

C.tangerine (Hongjv, CRJC32-01), C.junos(Tanaka),

C.aurantium(Poncirus trifoliata) were collected from

citrus germplasm resource nursery of Chongqing

Three Gorges Academy of Agricultural Sciences.

Resistance Identification Experiment. The

inoculation experiment was conducted in laboratory

of Chongqing Three Gorges Academy of Agricultural

Sciences in 2020-2021, following a modified method

of placing the mycelial plugs on the excised leaves

reported by Lin et al. (Lin, Huang, 2011).

15 separated leaves inoculated two sits on each

leaf, for 30 spots in total, were inoculated with a

typical strain WZBY1 in every variety. Leaves were

washed, air-dried, and surface-disinfested with 75%

ethanol using cotton swabs, and then put it into a

sterile tray covered with sterile gauze soaked with

distilled water. Subsequently, pricking ten times (but

not pierced) on the lower surface with an insect

needle. Two sclertiums (5 mm diameter) were placed

on the abaxial of each wounded leaf. For the

non-inoculated controls, leaves were put with the

same size of PDA only. All treatments were cultured

separately under 10 °C in a moisture box and

investigating the average diameter of the speckle and

incidence rate in in 28 days after inoculation. The

resistance was evaluated with the method of average

diameter of the speckle (Chen, Liu, 2009); (Hu, Liu,

2015); (Liu, Hu, 2013). The leaves were recorded as

infected if inoculated sits displaying disease

symptoms (Zhang, Ding, 2013); (Lin, Huang, 2011).

Assessment of the resistance grades of citrus

varieties to Ps. Citricarpa was according to Li et al.

(Li, Zheng, 2009). Incidence was obtained using the

formula: incidence (%) = (infected leaves/inoculated

leaves) × 100%.

All experiments were performed in triplicate. The

entire experiment was conducted triplicate.

Statistical Analysis. The database was analyzed by

IBM SPSS 16.0 (New York) and and Microsoft

Excel. The data are means of 30 replicates in

triplicate.

3 TEST RESULTS

3.1 Incidence Rate of Different Citrus

Cultivars

There were various degrees of differences on the

pathogenicity to test-cultivars, and even got

significant difference(P<0.05) among some

varieties. There was 8 varieties of incidence rate

were under 20%, 7 varieties among 21-50%, 2

varieties among 51-80%, 3 varieties displayed 100%

incidence. Newhall Navel Orange (C.sinensis), Orah

091 (C.reticulata) and CRIC32-01 (C.tangerine)

were got 100% incidence. The disease didn’t infect

Shatianyou, Hongbaoshiyou, Tarocco Blood Orange

No.8, Tanaka, Poncirus trifoliata. Incidence rate of

this 5 citrus cultivars were all 0%. Sanhongyou,

Daya mandarin and Taiguoqingyou displayed

3.33%, 6.67% and 20% for incidence. Incidence rate

of Newhall Navel Orange, CRIC32-01 and Orah 091

reached 100%, and Dianjiangwanyou,

DianjiangBaiyou and Liangpingyou were showed

40%-50%. The other varieties of C. grandis were

showed the incidence under 20% (Fig. 1, Fig. 2).

3.2 Diameter of Infection Sits of

Different Citrus Cultivars

Shatianyou, Hongbaoshiyou, Sanhongyou, Tarocco

Blood Orange No.8, Tanaka and Poncirus trifoliata

were 0mm in diameter of infection sits, and

ICBEB 2022 - The International Conference on Biomedical Engineering and Bioinformatics

398

displayed immune phenotype. Taiguoqingyou, and

Daya mandarin displayed 0.1m for average lesion

diameter, respectively. 6 varieties ranged from

0.2mm to 1.0mm for average diameters of lesions,

and 5 varieties were 1.1mm-5.0mm (Fig. 3).

In the 20 varieties, 18 (90 percent of all test

varieties) ranged from 0 to 3 mm in spots diameter

which can be classified into highly resistant varieties.

Orah 091 got 8.1mm for average lesion diameter of

which the maximal diameter could reach 14mm

which was defined as moderate resistant. CRIC32-01

gotting 3.8mm for average lesion diameter and 100%

for incidence which showed resistant to Ps.

Citricarpa. There was a particular example Newhall

Navel Orange, of which the average lesion diameter

got 1.8 mm but incidence was 100%, which was

identified with highly resistant variety according to

the diameter spots.

In C.grandis, except Liangpingyou, the other 6

test varieties were all classified to highly resistant. In

C.reticulata, Orha 091 was the most susceptible

variety(100% for the incidence, 8.1mm for the

diameter spots). Daya mandarin was classified to

highly resistant to Ps. Citricarpa according to the

diameter spots. In C.tangerine, incidence and

epidemic degree of CRIC32-01 was more severe,

gotting second diameter(3.8mm) in the test varieties

and reaching 100% for incidence. While the Hongjv

displayed significantly lower incidence (50%) of Ps.

Citricarpa (Fig. 2, Fig. 3), which was classified into

highly resistant group.

The results also suggested that the incidence rate

of varieties correlated linearly with lesion

diameter(R

=0.55) (Fig. 2). In all test objects, the

high incidence, such as CRIC32-01 and Orah 091,

existed a tendency to strong virulent and a high risk

to infected by Ps. Citricarpa in citrus production

areas in our country. In addition, for reducing the risk

of disease and economic losses, late-maturing,

suitable and strong resistant varieties should be

planted, including increasing Tarocco Blood Orange

No.8, Tango, Shiranui in upper and middle reaches of

Yangtze River. In western Hubei and western Hunan,

according to the current structure of citrus industry

and climatic conditions, the susceptible citrus, such

as Orah 091 should be appropriately reduced.

Depending on the natural advantages, Aiyuan No.38,

CRIC32-01 and Taiguoqingyou should be with due

consideration.

Figure 1: Symptoms of some citrus varieties after

inoculation.

A, Shatianyou; B, Hongbaoshiyou; C, Tarocco

Blood Orange No.8; D, Tanaka; E, Poncirus

trifoliata; F, Sanhongyou; G, Daya mandarin; H,

Taiguoqingyou; I, Tango; J,Shiranui;

K,Dianjiangwanyou; L, DianjiangBaiyou; M,

Aiyuan No.38; N, Tarocco Blood Orange No.9; O,

Liangpingyou; P, Hongjv; Q, Gold Nugget; R,

Newhall Navel Orange; S,CRIC32-01; T, Orah 091

Note: The data are means of 30 replicates. The experiment was conducted triplicate.

Figure 2: Correlational analyses of the incidence and diameter of infection sits different citrus varieties.

Evaluation of 20 Citrus Varieties Resistance to Pseudofabraea Citricarpa

399

Note: Columns represented means of 30 replicates, and bars represent standard deviation. The experiment was conducted

triplicate.

Figure 3: The incidence of different citrus varieties after inoculation.

4 DISCUSSIONS

Citrus target spot has become an important disease

infecting such as Satsuma mandarin, kumquat as

well as Eureka lemon et al. in some citrus-producing

regions reported in Shanxi Province (Zhu, Wang,

Huang, Zhang, Li, 2012) Chongqing City (Zhan,

2021), Hubei Province and Hunan Province (Xiao,

Zeng, Wang, Cheng, Li, 2020), China, which is

leading to substantial economic losses to citrus

production. Unlike the most diseases, the disease

infected by Ps. Citricarpa, prevails only in late

winter and early spring (Zhu, Wang, Huang, Zhang,

Li, 2012)

It causes defoliation, twig dieback,

fruit-dropping, and dramatically market value

reduction of infected fruits, especially on susceptible

citrus varieties. Furthermore, a high proportion of

trees become diseased or dead, and some orchards

have been destroyed.

Gene is the primary cause of resistance.

According to the characteristic of conserved domain

of resistance genes, sequence amplification of

conserved domain is a common method for

identification and discovery of resistance genes

(Fenillet, 1997), which has been applied to field

pepper (Zhang, Chen, 2008), soybean (Garzon,

2013), wheat (Xi, Wang, 2021) and other plants. The

key to study the resistance differences among citrus

germplasm, the resistance mechanism and even the

interaction between citrus and Ps. citricarpa is to

study the resistance genes of citrus. Study the

resistance genes was the basis of understanding the

resistance mechanism which needed to further

research.

5 CONCLUSIONS

Based on the results and discussions presented

above, the conclusions are obtained as below:

(1) Through the resistance evaluation of

dominant citrus varieties with the established

method, 18 varieties such as Shatianyou,

Hongbaoshiyou, Tarocco Blood Orange No.8,

Tanaka, Poncirus trifoliata, Sanhongyou, Daya

mandarin, Taiguoqingyou, Tango, Shiranui,

Dianjiangwanyou, DianjiangBaiyou, Aiyuan No.38,

Tarocco Blood Orange No.9, Liangpingyou, Hongjv,

Gold Nugget and Newhall Navel Orange were

classified into highly resistant varieties according to

the diameter spots. Only Orah 091 was defined as

moderate resistant. And CRIC32-01 was resistant

cultivars.

(2) According the results in this study, we

suggest increasing Tarocco Blood Orange No.8,

Tango in upper and middle reaches of Yangtze

River. In western Hubei and western Hunan, Orah

091 should be appropriately reduced. Aiyuan No.38,

CRIC32-01 and Taiguoqingyou should be with due

consideration.

ACKNOWLEDGMENTS

This research was funded by Science and

Technology Research Program of Chongqing

Municipal Education Commission (Grant

No.KJ202101254125241) and Chongqing Wanzhou

Science and Technology Program(wzstc-20210211).

ICBEB 2022 - The International Conference on Biomedical Engineering and Bioinformatics

400

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