UNIVERSIDADE ESTADUAL PAULISTA - UNESP 

CÂMPUS DE JABOTICABAL 

 

 

 

 

 

 

FLUSHING DYNAMICS AND 'Candidatus LIBERIBACTER 

ASIATICUS' MOVEMENT ON VARIOUS CITRUS SCION AND 

ROOTSTOCK COMBINATIONS 

 

 

Everton Vieira de Carvalho 

 Engenheiro Agrônomo 

 

 

 

 

 

 

 

2020 



UNIVERSIDADE ESTADUAL PAULISTA - UNESP 

CÂMPUS DE JABOTICABAL 

 

 

 

 

 

FLUSHING DYNAMICS AND 'Candidatus LIBERIBACTER 

ASIATICUS' MOVEMENT ON VARIOUS CITRUS SCION AND 

ROOTSTOCK COMBINATIONS 

 

 

 

 

Everton Vieira de Carvalho 

 

Orientador: Prof. Dr. Silvio Aparecido Lopes 

Coorientador: Dr. Eduardo Augusto Girardi 

 

 

Tese apresentada à Faculdade de Ciências 
Agrárias e Veterinárias - Unesp, Câmpus de 
Jaboticabal, como parte das exigências para 
obtenção do título de Doutor em Agronomia 
(Produção Vegetal). 

 

 

2020 



 

 

 

 

 

 

 

 

 

 

 

 

 

 



 

 

 

 



 

AUTHOR'S BIOGRAPHY 

 

Everton Vieira de Carvalho was born on August 24th, 1985, in Cruz das 

Almas municipality, Bahia State, Brazil. Everton is undergraduate in Agronomic 

Engineering (2014) from Federal University of Recôncavo da Bahia (UFRB). 

During his undergraduate studies, he had scholarship for two years in the 

concentration area of Plant Physiology and Seeds. In 2015, he initiated his 

Master studies at the same university, and finished in 2017, in the concentration 

area of Agricultural Sciences. He is currently a Ph.D. student in Agronomy (Crop 

Production) of the Faculty of Agricultural and Veterinary Sciences of the São 

Paulo State University Júlio Mesquita Filho (UNESP), in the concentration area 

of Pest and Disease Management and Control. He has experience in Agronomy 

with an emphasis on Citrus Industry, Plant Propagation of Citrus Plants and 

Phytopathology of Bacterial Diseases in Citrus. He undertook sandwich 

undergraduate at the University of Manitoba, in Winnipeg, Manitoba, Canada 

(2012/2013), in Agronomy, where he developed research in the area of Plant 

Physiology, Seed Technology and Molecular Biology.  

.  

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 



 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

..."don't lose the dreams inside your head"... 

Dave Matthews 

 



 

 

 

 

 

 

 

 

 

 

 

 

 

 

To my grandfather, Alderico Mendes 

Vieira, who lives forever in my 

memories and in my heart,  

I DEDICATE! 

 

To my mother, Auderice Lima Vieira, 

and my brother, Luiz Felipe Vieira de 

Carvalho, for all support and for 

always believing in me,  

I OFFER! 

 



 

ACKNOWLEDGMENTS 

 

To God, for all the blessings in my life. 

To my advisor, Dr. Silvio Aparecido Lopes, for all the conversations, 

teachings, trust, and friendship.  

To my co-advisor, Dr. Eduardo Augusto Girardi, for all teachings, 

encouragement, trust, and friendship. 

To all examiners that composed the qualification and thesis defense 

committee: Dr. Andrew Beattie, Dr. Georgios Vidalakis, Dr. Jim Graham, Dr. Ute 

Albrecht and Dr.Glauco Rolim. Thank you so much for all considerations that will 

help us to improve this research. 

To the São Paulo State University - Faculty of Agricultural and Veterinary 

Sciences – UNESP/FCAV, Jaboticabal-SP, Faculty Staff and friends, on behalf 

of the head department of the Graduate Program in Agronomy (Crop Production), 

Prof. Dr. Rouverson Pereira da Silva, for the teachings and for the opportunity to 

do the doctorate degree.  

To the employees of the Graduate Technical Session for all their 

dedication and commitment. 

To Fundecitrus and all staff in the Research and Development department 

and the diagnosis laboratory, on behalf of Mr. Juliano Ayres. 

To my research teammates and friends: Fernanda, Hermes, Luis, Priscila, 

and Wellington, for all support on laboratory and field experiments. Thank you 

very much for all support in conducting this research and friendship. Also, the 

interns Rafaela and Abner, for all support on greenhouse experiments. 

To Laudecir for all partnership in my graduate studies. Thank you so much 

for all support in this entire research; the experiments, assessments, writing and 

friendship. 

To Juan, for the friendship and support in the experiments and in the thesis 

review. 

Also, for all friends that I have made on this journey. All interns and 

employees, especially Mônica, Rafael and Samuel. 

To all greenhouse staff at Fundecitrus: André, Anélio, Eliabe, José 

Rodrigues, Leomar and Miguel, for all support. 



 

The Experimental Station of Citriculture in Bebedouro, on behalf of Mr. 

Otávio Sempionato. To Dr. Eduardo Sanches Stuchi, for the friendship all these 

years in my graduate studies. And to the employees, especially Patrick, Élio, and 

Gustavo (Guru) for their support and friendship. 

To my family: my grandfather Alderico (in memorium), my grandmother 

Maria Áurea; my uncles Elvis and Grécio; my cousins Rodrigo, Catarina, 

Gabriela, Pedro, and Helena. To my mother Auderice and my father Luis Jacson; 

my brothers Felipe, Maria Helena, Junior and Maria Luiza. And to my beloved 

nephews, the little ones Tiago and Rafael. For all the love and missing. 

 To my beloved girlfriend, Stefany Souza. Thank you so much for all the 

happiness that you have brought into my life. For all the love, care, and complicity. 

I love you! 

To my friends from Jaboticabal, especially from TOca Fogo’s fraternity: 

Adão Santos, Edgard Silva, Emmanuel Moreira, Luan Oliveira, Antonio Tassio 

and Renato Soares. Also, my dear friends Taynara Valeriano, Larissa Nogueira, 

Maria Albertina, and Kárita Almeida. Thank you, guys, for all the good moments 

in my graduate life. 

To my friends from my hometown, that even though they were distant, their 

support always made me feel close to them. Also, to all my friends from 

Araraquara. 

For the Brazilian National Council for Scientific and Technological 

Development (CNPq), for the doctorate scholarship. 

This work was carried out with the support of Coordenação de 

Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Código de 

Financiamento 001.  
Finally, to everyone who somehow helped me achieve these graduate 

studies, THANK YOU SO MUCH! 

 

 

 

 

 

 



i 
 

SUMMARY 
 

RESUMO............................................................................................................ iii 

ABSTRACT ........................................................................................................ iv 

CHAPTER 1 – General considerations .............................................................. 1 

1. INTRODUCTION ......................................................................................... 1 

2. LITERATURE REVIEW ............................................................................... 3 

2.1. Socioeconomic importance of the citrus industry to Brazil ........................ 3 

2.2. Commercial scion and rootstock varieties in Brazil ................................... 4 

2.3 Citrus flushing dynamics ............................................................................... 7 

2.4 Huanglongbing: historical background and management ............................. 8 

2.5 Candidatus Liberibacter spp. and insect vectors .......................................... 9 

2.6 Rootstock responses to HLB ...................................................................... 11 

2.7 Translocation of Candidatus Liberibacter spp. ........................................... 12 

3. REFERENCES .......................................................................................... 13 

CHAPTER 2 - Modeling seasonal flushing and shoot growth on different citrus 

scion-rootstocks ............................................................................................... 23 

1. INTRODUCTION ....................................................................................... 24 

2. MATERIAL AND METHODS ........................................................................ 26 

2.1 Experimental area and plant material ......................................................... 26 

2.2 Flushing dynamics and intensity ................................................................. 27 

2.3 New shoot growth dynamics ....................................................................... 28 

2.4 Data analysis .............................................................................................. 29 

2.4.1 Flushing dynamics and intensity .............................................................. 29 

2.4.2 New shoot growth .................................................................................... 29 

3. RESULTS ..................................................................................................... 30 

3.1 Flushing dynamics and intensity ................................................................. 30 

3.2 Relationship between flushing and environmental conditions .................... 38 

3.3 Relationship between new shoot growth and environmental conditions ..... 41 

4. DISCUSSION ............................................................................................... 43 

5. REFERENCES .......................................................................................... 46 

CHAPTER 3 – Impacts of trifoliate and non-trifoliate hybrid rootstocks on 

‘Candidatus Liberibacter asiaticus’ movement within citrus trees ..................... 54 



ii 
 

1. INTRODUCTION ....................................................................................... 55 

2. MATERIAL AND METHODS ........................................................................ 57 

2.1 Las movement in ‘Rangpur’ lime and ‘Flying Dragon’ rootstocks ............... 57 

2.2 qPCR analysis ............................................................................................ 59 

2.3 Data analysis .............................................................................................. 59 

2.4 Las movement in various rootstock-scion combinations............................. 60 

2.5 Data analysis .............................................................................................. 61 

3. RESULTS ..................................................................................................... 62 

3.1 Las movement in ‘Rangpur’ lime and ‘Flying Dragon’ rootstocks ............... 62 

3.2 Las movement in additional scion-rootstock combinations ......................... 64 

4. DISCUSSION ............................................................................................... 71 

5. REFERENCES ............................................................................................. 73 

CHAPTER 4 – Final considerations ................................................................. 81 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 



iii 
 

DINÂMICA DE BROTAÇÃO E MOVIMENTAÇÃO DE 'Candidatus 
LIBERIBACTER ASIATICUS' EM COMBINAÇÕES DE COPA E PORTA-

ENXERTO DE CITROS 
 
 

RESUMO - ‘Candidatus Liberibacter asiaticus’ (Las) é a bactéria 
associada ao huanglongbing (HLB), transmitida pelo psilídeo asiático Diaphorina 
citri. D. citri é atraído por brotações novas (BN), onde ocorre preferencialmente 
a aquisição e transmissão da bactéria. A maioria dos estudos envolveram 
variedades com níveis similares de vigor. Ainda, pouco se sabe sobre o processo 
de infecção e colonização de Las. Este estudo foi conduzido para avaliar 
combinações de copa e porta-enxerto na dinâmica e intensidade de brotações e 
a movimentação de Las. No campo, as laranjeiras ‘Pera’ e ‘Folha Murcha’, a 
tangerineira ‘Ponkan’ e a limeira ácida ‘Tahiti’, em nove porta-enxertos, foram 
avaliadas. As BN foram contadas e classificadas a cada 20 dias. O número de 
BN e o cálculo da área abaixo da curva da dinâmica de brotações foram 
utilizados para comparar a intensidade de BN. Regressão linear múltipla foi 
utilizada para descrever as ocorrências das BN. Máximas, médias e mínimas 
temperaturas diárias e a chuva acumulada como variáveis independentes. O 
crescimento das BN foi avaliado durante a primavera e o outono com regressões 
não-lineares em função de graus-dia. As ocorrências das BN foram similares, 
mas a intensidade foi maior no limoeiro ‘Rugoso’. As ocorrências em ‘Pera’, 
‘Folha Murcha’ e ‘Tahiti’ associaram-se positivamente com o aumento das 
temperaturas mínima e média, enquanto para a ‘Ponkan’, com a chuva 
acumulada. As BN cresceram mais rápido e atingiram maiores comprimentos na 
primavera, exceto para a ‘Folha Murcha’. Os valores do R2 ajustado encontrados 
para o crescimento das BN podem auxiliar e ajustar o tempo e a frequência das 
aplicações de inseticidas. Em casa-de-vegetação, um método para avaliar o 
impacto do porta-enxerto na movimentação de Las foi proposto. Foi realizada 
uma remoção vertical do tecido da casca do caule principal das plantas. Plantas 
de ‘Valencia’ enxertadas em limoeiro ‘Cravo’ ou trifoliata ‘Flying Dragon’ foram 
utilizadas. As plantas foram inoculadas em um dos ramos, e o ramo oposto e as 
raízes foram avaliados a cada 15 dias. A movimentação de Las, título bacteriano 
e perda de raiz foram avaliados. O movimento ascendente de Las e a perda de 
raiz foram maiores no ‘Cravo’, enquanto o descendente e o título bacteriano no 
caule e nas raízes foram similares. A movimentação ascendente de Las se 
correlacionou positivamente com o crescimento de BN, e o título bacteriano no 
caule ou na raiz com a perda de raiz apenas para o ‘Cravo’. O método foi utilizado 
para estudar combinações de ‘Pera’, ‘Folha Murcha’, ‘Ponkan’ e ‘Tahiti’ em 16 
porta-enxertos. Híbridos de trifoliata induziram menor movimentação de Las, 
título bacteriano e perda de raiz. A análise de componentes principais confirmou 
a associação entre a movimentação de Las e o crescimento de BN, e título 
bacteriano e perda de raiz. A análise de Cluster dividiu os porta-enxertos em dois 
grupos, independentes da natureza genética. Não houve porta-enxerto que 
interagiu de modo similar com todas as copas, indicando que o impacto do HLB 
variou de acordo com a combinação de copa e porta-enxerto. 
 
Palavras-chave: Citrus spp., Poncirus trifoliata, estádios fenológicos, 
crescimento, condições meteorológicas, modelagem, porta-enxertos, 
movimentação de Liberibacter. 



iv 
 

FLUSHING DYNAMICS AND 'Candidatus LIBERIBACTER ASIATICUS' 
MOVEMENT ON VARIOUS CITRUS SCION AND ROOTSTOCK 

COMBINATIONS 
 
 

ABSTRACT - ‘Candidatus Liberibacter asiaticus’ (Las) is a phloem-limited 
bacterium associated with huanglongbing (HLB), transmitted by the Asian citrus 
psyllid (ACP) Diaphorina citri. ACP is attracted by new shoots (NS), in which 
preferably occurs feeding, reproduction, nymph development, bacterial 
acquisition and transmission. Most studies about flushing have involved citrus 
varieties with similar growth vigor levels. Also, little is known about infection and 
colonization by Las. This study was conducted to evaluate whether scion and 
rootstock combinations would affect flushing dynamics and intensity and Las 
movement in citrus trees. In the field, ‘Pera’ and ‘Folha Murcha’ sweet oranges, 
‘Ponkan’ mandarin and ‘Persian’ lime were growing on nine rootstocks. NS were 
counted and classified every 20 days. The number of NS and the area under flush 
shoot dynamics were used to compare NS intensities. Multiple regression was 
used to describe NS occurrences. Maximum, minimum and average 
temperatures and accumulated rainfall were used as independent variables. NS 
growth was evaluated during the spring and autumn and data compared with non-
linear regression based on degree-days. NS occurrences were similar, but 
intensity was higher on ‘Rough’ lemon than ‘Flying Dragon’. NS occurrences on 
‘Pera’, ‘Folha Murcha’ and ‘Persian’ lime were associated positively with an 
increase of minimum and average temperatures, while on ‘Ponkan’ with 
accumulated rainfall. NS grew faster and attained longer lengths during spring 
than in autumn, except for ‘Folha Murcha’. Adjusted R2 attained for NS growth 
can be used to improve timing and adjust frequency of insecticide applications. 
In the greenhouse, a method to assess the impact of the rootstock on the 
bacterial movement is proposed. A vertical bark strip removal was performed in 
the main stem of the trees. 1.5-year-old potted trees of ‘Valencia Late’ growing 
on ‘Rangpur’ lime or ‘Flying Dragon’ trifoliate were used. All trees were graft-
inoculated on one branch and the opposite branch and the roots were assessed 
fortnightly by qPCR. Data for Las movement, bacterial titer and root loss were 
evaluated. The upward Las movement and root loss were higher on ‘Rangpur’ 
lime, while the downward Las movement and titer on both shoots and roots were 
similar. Las movement upward positively correlated with new shoot growth. 
Positive correlation was observed between titer in the shoot or roots and root loss 
only for ‘Rangpur’ lime. The method was used in a study of 2.5-year-old potted 
trees of ‘Pera’, ‘Folha Murcha’, ‘Ponkan’ and ‘Persian’ lime growing on 16 
rootstocks. The same variables were evaluated, and inoculations performed as 
described above. The opposite branch was assessed monthly. Trifoliate hybrids 
rootstocks induced lower Las movement, titer, and root loss. Principal component 
analysis confirmed association between Las movement and new shoot growth, 
and titer and root loss. Cluster analysis separated rootstocks into two groups 
regardless the genetic background. There was no rootstock that interacted 
similarly with all scions, indicating that HLB impact varies according to scion-
rootstock combination. 
 
Keywords: Citrus spp., Poncirus trifoliata, phenological stages, tree growth, 
meteorological conditions, modeling, rootstocks, Liberibacter movement. 



1 
 

CHAPTER 1 – General considerations 

 

1. INTRODUCTION 

 

Sweet oranges are the most cultivated citrus trees in the world, accounting 

for ~ 60% of the global production. Brazil is the largest sweet orange producer 

worldwide, accounting for around 18.6 million tons annually. China is the largest 

mandarin producer, with an approximate production of 19 million tons. For 

lemons, Argentina is the largest producer worldwide, accounting for ~ 25% of the 

global production, and the main lime-producing countries are Mexico and India 

(FAO, 2020). 

The area cultivated with sweet oranges in Brazil comprised 654.3 

thousand hectares (ha) in the 2019/2020 forecast. Approximately 424.8 thousand 

ha from the total planting area is located in the State of São Paulo (SPS) and in 

west-southwest of Minas Gerais, representing the most extensive citrus belt in 

Brazil (Fundecitrus, 2020a; IBGE, 2020). However, several phytosanitary 

problems associated with the occurrence of pests and diseases have 

endangered the sustainability of the Brazilian citrus industry (Mattos Junior et al., 

2005). Among the diseases that harm citrus crops, huanglongbing (HLB) is the 

most severe due to the susceptibility of all citrus varieties leading to high yield 

losses (Bové, 2006). 

HLB is associated with Gram-negative, phloem-limited bacteria 

‘Candidatus Liberibacter africanus’ (Laf), ‘Ca. L. asiaticus’ (Las) and ‘Ca. L. 

americanus’ (Lam) (Bové, 2006; Teixeira et al., 2005). Las and Lam are naturally 

spread in the field by the Asian citrus psyllid (ACP) Diaphorina citri Kuwayama 

(Hemiptera: Sternorrhyncha: Psylloidea) (Capoor et al.,1967; Yamamoto et al., 

2006), while Laf is spread by the African citrus psyllid Trioza erytreae (Del 

Guercio) (Hemiptera: Psyllidae) (McClean and Oberholzer, 1965). ACP is highly 

attracted by new shoots of citrus trees, on which they preferably feed and 

reproduce (Cifuentes-Arenas et al., 2018; Grafton-Cardwell et al., 2013). New 

shoots are the tissues from which Las is acquired by ACP and to which the 

bacterium is transmitted (Hall et al., 2016; Sétamou et al., 2016). Since the 

population dynamics of ACP is consistently associated with the presence of new 

shoots in citrus trees (Yamamoto et al., 2001; Tsai et al., 2002; Kistner et al., 



2 
 

2016; Teck et al., 2011), understanding the dynamics of flushing occurrence 

helps to improve HLB management. Understanding the dynamics of growth and 

development of the new shoots also is important, since the efficacy of chemical 

ACP control decreases as they grow and develop (De Carli et al., 2018).  

After transmission, the bacterium will colonize all tissues except the seed 

endosperm (Tatineni et al., 2008; Li et al., 2009). Recent studies have 

demonstrated how cell-to-cell movement of Las takes place within the phloem 

(Achor et al., 2020). In longer distance, the bacterium moves predominantly 

towards new tissue growth, either on branches or roots (Raiol-Junior et al., 2020). 

The roots seem to play an important role in the infection process of the whole 

tree, being an important site for pathogen establishment (Johnson et al., 2014; 

Raiol-Junior et al., 2020).  

Several studies have shown that some rootstocks are less affected by Las 

infection, mainly hybrids having Poncirus trifoliata (L.) Raf as a parent. Milder 

rootstock responses to infection include lower bacterial titers, and less severe 

symptoms in the canopy (Boava et al., 2017; Albrecht et al., 2012; Albrecht and 

Bowman, 2012; Folimonova et al., 2009). These studies have contributed to a 

better understanding of the importance of the root system for survival of the tree 

during pathogen attack. A rootstock that delays colonization or reduces Las 

multiplication would aid in minimizing the impacts of the disease in the field. 

Since the rootstock induces changes in several characteristics, such as 

growth and vigor of the canopy (Pompeu Junior, 2005), such changes may alter 

the attractiveness to ACP and potentially the dynamics of bacterial translocation 

in tree tissues. Promising rootstocks have been evaluated regarding mainly fruit 

production, tolerance to water deficit and tree size. However, they were not 

investigated regarding their influence on flush shoot dynamics of the scion 

variety, as wells as on maturation of new shoots over the seasons. These are 

important aspects to determine best time and frequencies of insecticide 

application. In addition, nothing is known with respect to the responses of these 

new rootstocks to Las infection. Thus, this information is important to predict 

performance against HLB when these rootstocks become fully available and are 

planted on a large scale.  

This study was conducted with the objectives of evaluating a range of 

several commercial and non-commercial rootstock hybrids currently under field 



3 
 

investigation in Brazil (i) in terms of phenological changes induced to the scion, 

and how the changes could potentially impact ACP reproduction and the 

effectiveness of ACP chemical control, and (ii) in terms of changes in the 

magnitude of Las movement and colonization of tree tissues, and magnitude of 

damage to the root system, making the trees more or less tolerant to the disease. 

 

2. LITERATURE REVIEW 

 

2.1. Socioeconomic importance of the citrus industry to Brazil 

 

Plants of the genus Citrus and related genera (Fortunella and Poncirus) 

belong to botanical family Rutaceae, subfamily Aurantioideae, and are originated 

mostly from Southeast Asia (Swingle and Reece, 1967; Scora, 1975). The 

cultivated citrus resulted from complex crosses between citron (Citrus medica L.), 

pummelo (Citrus maxima [Burm.] Merrill), mandarin (Citrus reticulata Blanco) and 

Citrus micrantha Wester (Wu et al., 2018). In Brazil, the seeds for propagation of 

the first citrus trees were brought to the country by the Portuguese, during the 

colonization period that started in 1530. First reports of citrus plantings were in 

São Paulo state (SPS) which later expanded along the coast and to other regions 

in Brazil (Hasse, 1987). 

In the 2019/2020 national forecast, SPS reached a total productivity of 

386.7 million boxes of 40.8 kg of sweet orange (Citrus × aurantium var. sinensis 

L.), followed by the states of Minas Gerais (24.0 million), Paraná (19.5 million), 

Bahia (19.3 million) and Sergipe (9.2 million). The estimated forecast for 

2020/2021 accounts for 286.7 million boxes of 40.8 kg only for the citrus belt of 

SPS and west-southwest Minas Gerais (Agrianual, 2020; Fundecitrus, 2020a). In 

addition to sweet orange, Brazil is an important producer of mandarin and its 

hybrids, lemons [C × limon var. (L.) Burm. f.], and acid limes (C. × latifolia var. 

latifolia). Brazil is the major producer of frozen and concentrated orange juice, the 

second largest producer of concentrated tangerine juice (behind only the United 

States), and the fourth largest producer of concentrated lemon juice, with the 

leader in world production being Argentina (FAO, 2020). As a result, the Brazilian 

citrus industry is one of the most important sources of taxes and jobs, 

encompassing nurseries, farms, suppliers, fresh fruit commerce, and juice 



4 
 

processing and essential oil industries among other segments. In 2019, the citrus 

industry generated over 48,000 jobs in SPS, which represented 26.2% of the total 

jobs generated by all sectors of the state economy (CitrusBr, 2020). 

 

2.2. Commercial scion and rootstock varieties in Brazil 

 

Sweet oranges, mandarins and acid limes represent the most cultivated 

citrus scion varieties in Brazil (Pio et al., 2005). In the last tree inventory survey 

of bearing trees conducted by Fundecitrus (2020a), the total area of the citrus 

belt of SPS and west-southwest of Minas Gerais was comprised by 18.5% with 

the early-season sweet orange varieties ‘Hamlin’, ‘Westin’, ‘Rubi’ and ‘Valencia 

Americana’, 35% with the midseason varieties, mainly ‘Pera’, and 42% with the 

late-season ‘Valencia Late’, ‘Folha Murcha’ and ‘Natal’. Lemons and acid limes 

corresponded to 8.4% of the total bearing trees, of which 90% were of ‘Persian’ 

lime and 9% within the true lemon group, mainly the Sicilian lemon. Mandarins 

comprised 2.6% of the bearing trees and were represented mainly by ‘Ponkan’ 

(Citrus reticulata Blanco) and ‘Murcott’ tangor (C. × sinensis x C. reticulata) 

(Fundecitrus, 2020a). 

‘Pera’ is the most important sweet orange, characterized by vigorous trees 

with excellent productivity. Fruits are suitable for fresh market and industrial 

processing. ‘Valencia Late’ ranks second with exceptional productivity. Fruits are 

larger than those of ‘Pera’ and are suitable for fresh market and especially for 

industrial processing. This variety is compatible with all commercial rootstock 

varieties used in Brazil, while ‘Pera’ is incompatible with some rootstocks (Barry 

et al., 2020; Figueiredo, 1991; Pio et al., 2005).  

‘Folha Murcha’ has the remarkable characteristic of curled leaves that 

resemble wilting due to water deficit. It maturates later than ‘Valencia Late’, and 

the trees are less vigorous than those of ‘Pera’. Among other scion varieties of 

economic importance comes the highly productive 'Hamlin' with fruits suitable 

mainly for industrial processing. ‘Natal’ produces high quality fruits suitable for 

fresh market and industrial processing (Barry et al., 2020; Figueiredo, 1991; Pio 

et al., 2005).  

Mandarin is one of the original progenitors of all modern citrus varieties. 

The mandarin group is highly diverse. ‘Ponkan’ is characterized by trees with 



5 
 

compact and upright growth habit. It is an early-midseason variety with irregular 

and biennial bearing. Fruits are large, quite easy to peel, with a mild sweet flavor, 

which makes ‘Ponkan’ highly appreciated for fresh consumption in Brazil (Barry 

et al., 2020; Pio et al., 2005).  

As the other citrus, the acid limes and lemons are usually well-adapted to 

tropical and subtropical climates. The trees are characterized as having multiple 

blooms and crops of fruit. ‘Eureka’ is the lemon variety most cultivated in Brazil. 

This variety is precocious with high production in the early years after planting. 

‘Eureka’ fruit tend to be smaller than the other important lemon varieties, but they 

have high juice content and are very suitable for fresh market. In relation to acid 

limes, the clones ‘IAC-5’ and ‘Quebra-Galho’ are the most cultivated in SPS. ‘IAC-

5’ is a nucellar clone with high productivity and tolerance levels to CTV (Citrus 

tristeza virus, CTV). Trees are vigorous and an orchard of this clone is highly 

uniform. ‘Quebra-galho’ is an old clone that was infected with citrus viroids, 

including the citrus exocortis viroid (CEVd), which affects growth habit and 

reduces tree size (Barry et al., 2020; Figueiredo, 1991; Pio et al., 2005; Vidalakis 

et al., 2011).  

Rootstocks are responsible for inducing changes on the scion variety in 

several characteristics, such as vegetative growth, precocity of fruit production, 

yield, fruit maturation and quality, and tolerance to drought, diseases, and pests. 

Therefore, it is important to diversify the use of rootstocks, in order to better 

support the different scion varieties and adapt to biotic and abiotic stresses 

(Pompeu Junior, 2005). 

In 2016 the most cultivated rootstock varieties in SPS were ‘Swingle’ 

citrumelo [C. × aurantium var. paradisi ined. x Poncirus trifoliata (L.) Raf.] (50% 

of trees grafted in nurseries), ‘Rangpur’ lime (C. × limonia Osbeck var. limonia) 

(33%), ‘Sunki’ (C. reticulata ‘Sunki’) (11.9%) and ‘Cleopatra’ mandarins (C. 

reticulata ‘Reshni’) (1.2%) and trifoliate orange (P. trifoliata) (3.8%) (Carvalho et 

al., 2019).  

In Brazil, citrus have been propagated by grafting since the early twentieth 

century. At the beginning, the ‘Caipira’ sweet orange was the only rootstock used. 

Because of its susceptibility to drought and Phytophthora gummosis, it was 

replaced by sour orange (C. × aurantium L. var. aurantium). However, due to the 

onset of citrus tristeza virus (CTV) in the 1930’s, sour orange was replaced by 



6 
 

the ‘Rangpur’ lime (Chapot, 1975; Pompeu Junior, 2005). From the 1960s, 

‘Rangpur’ lime has become the major rootstock in citrus orchards in SPS. Besides 

the tolerance to CTV, trees on this rootstock are very vigorous and productive, 

similar to ‘Rough’ lemon (C. × limonia var. jambhiri ined.). Trees typically bear 

fruit at an early stage and are highly tolerant to drought (Pompeu Junior, 2001). 

Citrus blight started to affect trees grafted on ‘Rangpur’ lime in the 1970s and 

caused a diversification in the use of rootstocks. Then, ‘Cleopatra’ and ‘Sunki’ 

mandarins, and ‘Swingle’ citrumelo started to be planted in the orchards in the 

1990s. However, ‘Rangpur’ lime still predominated in the new plantings until 

1999, with the first reports of citrus sudden death (CSD) that affected scion 

varieties grafted on this rootstock (Rodriguez et al., 1979; Gimenes-Fernandes 

and Bassanezi, 2001).  

In 1907, ‘Swingle’ citrumelo was created in Florida by Walter T. Swingle. 

It was introduced into Brazil in 1948. It started to be more used in the 1980s, due 

to its tolerance to CSD, blight and gummosis. It is less tolerant to drought than 

‘Rangpur’ lime and is graft compatible with ‘Pera’ sweet orange, ‘Murcott’ tangor 

and ‘Eureka’ lemon (Hutchison, 1974; Pompeu Junior, 2005). Considered native 

from China, ‘Sunki’ mandarin started to be used in Brazil in 1985. ‘Sunki’ is 

reported to be tolerant of CTV, blight, but susceptible to Phytophthora. Fruit yield 

and quality are generally superior to trees on ‘Cleopatra’ (Pompeu Junior, 2005; 

Pompeu Junior et al., 2008).  

Trifoliate orange is regarded as a separate genus from citrus. Many 

different selections of this species exist. In general, trees on trifoliate orange have 

a low to moderate vigor but bear a good crop of fruit for the tree size. They are 

noted to be resistant or tolerant to some Phytophthora species, CTV, and cold, 

but sensitive to drought. Trifoliate orange rootstocks seem more favorably suited 

to areas with cool winter temperatures and are often regarded as a good choice 

where significant tree size control is desired, especially the variety ‘Flying Dragon’ 

(P. trifoliata var monstrosa) (Pompeu Junior, 2005; Stuchi et al., 2012).  

Constituting a new generation of rootstocks, citrandarins are hybrids 

resulting from the cross between mandarins such as 'Sunki' or 'Cleopatra' with 

trifoliate oranges. They possess characteristics inherited from the parents, in 

addition to the dwarfing effect in some hybrids (Pompeu Junior et al., 2002; 

Blumer and Pompeu Junior, 2005; Pompeu Junior and Blumer, 2014). Other 



7 
 

rootstock hybrids have been evaluated and have shown promising results, with 

potential for commercial use. Studies carried out in São Paulo showed that the 

hybrids ‘Sunki’ × (‘Rangpur’ lime × P. trifoliata) – 059 and (‘Rangpur’ lime × P. 

trifoliata) - 001 as rootstocks for 'Valencia Late' induced high productivity and 

drought tolerance, in addition to a reduced canopy volume. The hybrid ‘Sunki’ × 

‘Swingle’ – 041 have combined high productivity with a dwarfing effect compared 

to standard rootstocks, such as the ‘Rangpur’ lime and the ‘Sunki’ mandarin 

(Ramos et al., 2015). 

 

2.3 Citrus flushing dynamics  

 

Vegetative growth in citrus trees occurs in seasonal cycles or flushes 

throughout the year. Lemons and acid limes develop new shoots all year-round, 

while in sweet oranges they are seasonally defined (Spiegel-Roy and 

Goldschmidt, 1996). Flushing is normally more intense during the spring-

summer, less intense during early autumn, and absent in winter (Primo-Millo and 

Agustí, 2020). Air temperature and soil moisture are the main environmental 

components that regulate flushing (Primo-Millo and Agustí, 2020; Davies and 

Albrigo, 1994). Spring flush contains both vegetative and reproductive shoots. 

The midsummer and subsequent flushes are generally vegetative (Spiegel-Roy 

and Goldschmidt, 1996).  

New shoot growth also is governed by the genetic nature of the rootstock 

and scion. Rootstocks are widely used to control tree size and vigor, thus 

affecting the vegetative growth (Bowman and Joubert, 2020; Spiegel-Roy and 

Goldschmidt, 1996). Rootstock influence is governed by physiological 

mechanisms. Auxin accumulation, for instance, is generally greater in vigorous 

rootstocks and affects vascular differentiation, stimulating cambial activity and 

xylem development (Soumelidou et al., 1994; Ahn et al., 1999). Hence, higher 

hydraulic conductivity will favor nutrient uptake, contributing to vegetative growth 

(Atkinson et al., 2003; Olmstead et al., 2006).  

Knowledge on the impact of the rootstock on the canopy phenology is 

important. It may be used to program planting density of new orchards, and the 

subsequent cultural practices such as pruning and harvesting, and to improve 

pest management programs (Forner-Giner et al., 2014). However, most efforts 



8 
 

toward understanding flushing dynamics of citrus to improve ACP control and, 

consequently, HLB management, have involved rootstock and scion varieties 

with similar levels of growth vigor (Garieri, 2016; Oliveira, 2017).  

Since ACP is more attracted to citrus trees for feeding and reproduction 

during flushing periods (Yamamoto et al., 2001; Kistner et al., 2016), a less 

vigorous rootstock would be less attractive to citrus pests. For example, the 

dwarfing rootstock ‘Flying Dragon’ trifoliate induced lower canopy volume for 

‘Valencia Late’ sweet orange and a lower cumulative HLB incidence compared 

to the vigorous ‘Rangpur’ lime in an orchard under ACP control (Rodrigues, 

2018). 

 

2.4 Huanglongbing: historical background and management  

 

Huanglongbing (HLB) was first reported in 1919 in southern China as 

yellow shoot disease (Reinking, 1919). In 1928, a disease similar to HLB was 

reported in South Africa, under the name yellow shoot, while the name greening 

prevailed later (Merwe and Andersen, 1937). In Philippines, the disease was 

known as mottle leaf, in India as dieback, in Indonesia as vein phloem 

degeneration, and in Taiwan as likubin (Bové, 2006).  

HLB affects three of the major citrus producers in the world: China since 

1920’s; Brazil since 2004; and the United Sates since 2005 (Coletta-Filho et al., 

2004; Halbert, 2005; Teixeira et al., 2005; Bové, 2006). HLB was already reported 

in other Latin America countries, such as Cuba in 2007, (Luis et al., 2009), 

Dominican Republic in 2008 (Matos et al., 2009), Mexico, Belize, Jamaica and 

Puerto Rico in 2009 (Hall et al., 2013), Costa Rica in 2011 (Molina-Bravo et al., 

2015), Paraguay in 2013 (Senave, 2013), Argentina in 2012 (Agostini et al., 2015) 

and Colombia in 2015 (Ica, 2015). 

In Brazil, after the first report, the disease spread to all regions in SPS, 

with higher incidence in the center, reaching in 2020 an average 20.9 % of total 

incidence in the citrus belt of SPS and west-southwest Minas Gerais 

(Fundecitrus, 2020b). In Minas Gerais State, the first HLB outbreak was reported 

in 2005, in the southern region of the state (Castro et al., 2010). In 2019, HLB 

was already present in 60 municipalities of southern Minas Gerais and southern 

and southwestern Triângulo Mineiro area (Instituto Mineiro de Agropecuária, 



9 
 

2019). In Paraná State, HLB was first reported in 2007, in the northwest (Nunes 

et al., 2010) and, in 2019, HLB was present in 138 municipalities in the northern, 

northwestern, and western regions (Agência de Defesa Agropecuária do Paraná, 

2019). In August 2019, the disease was reported in 16 municipalities of the State 

of Mato Grosso do Sul (Diário Oficial da União, 2019). 

Identification of the disease is made by observation of symptoms, namely, 

yellow shoots, leaf blotchy mottle, lopsided fruits, and aborted seeds (Bové, 

2006). Symptoms are pronounced in autumn and winter seasons. Higher 

symptom expression in these seasons is due to the lower temperatures, which 

favor bacteria multiplication in plant tissues (Lopes et al., 2009a; Bassanezi et 

al., 2010). 

HLB management comprises planting of healthy trees from protected and 

certified nurseries, identification, and eradication of symptomatic trees to reduce 

sources of inoculum in the farm and neighboring areas, and psyllid monitoring 

and control (Belasque Jr et al., 2010; Bassanezi et al., 2020). In addition, HLB 

management must be applied in a regional area-wide scale. Thus, all neighboring 

orchards in the same region should carry out control measures, aimed at 

elimination of external inoculum sources and uniform control of the psyllid 

(Bassanezi et al., 2010; Bassanezi et al., 2020). 

 

2.5 Candidatus Liberibacter spp. and insect vectors 

 

The phloem-limited, Gram-negative, -Proteobacteria associated with 

HLB are ‘Candidatus Liberibacter asiaticus’ (Las), ‘Ca. L. africanus’ (Laf) and ‘Ca. 

L. americanus’ (Lam) (Garnier et al., 1984; Da Graça, 1991; Jagoueix et al., 1994; 

Bové, 2006). Las, the most widespread, is present in Asia, Africa, and Americas 

(Bové, 2006). Laf occurs in Africa (Bové, 2006) while Lam has been reported only 

in Brazil (Teixeira et al., 2005). 

Initially Lam was the most common HLB agent in Brazil. However, Las 

became the predominant species in São Paulo orchards (Lopes et al., 2009a). 

Currently, Las is present in over 99.9% of all positive field samples analyzed at 

the Fundecitrus’ diagnostic laboratory. This change in prevalence seems to be 

associated with Las tolerance to higher temperatures along with the higher titers 

reached by Las in plant tissues, which favors acquisition, transmission and 



10 
 

spread by the insect vectors in distinct citrus growing locations (Lopes et al., 

2009a; b; 2013; 2017; Gasparoto et al., 2012). 

Las and Lam are transmitted by the Asian citrus psyllid (ACP) Diaphorina 

citri Kuwayama (Hemiptera: Sternorrhyncha: Psylloidea), present in Asia, Africa, 

and Americas. Laf is transmitted by Trioza erytreae (Del Guercio) (Psyllidae), 

present in Africa, southwestern Europe (Portugal and Spain), the Arabian 

Peninsula (Saudi Arabia and Yemen), and in Madeira and Canary Islands (Bové, 

2006; Shimwela et al., 2016; Eppo, 2020). 

ACP was first reported in Brazil in the 1940’s (Costa Lima, 1942). ACP is 

highly attracted to new shoots of citrus trees, on which it preferably feeds, 

reproduces, develops (Cifuentes-Arenas et al., 2018; Grafton-Cardwell et al., 

2013), and acquires and transmits the pathogen (Hall et al., 2016; Sétamou et 

al., 2016). Females lay 500 to 800 eggs during their life cycle. Nymphs develop 

on shoots from early vegetative stages (Husain and Nath, 1927; Tsai and Liu, 

2000; Nava et al., 2007).  

The psyllid’s life cycle includes an egg stage and five nymphal instars. ACP 

adults are small (2.7 to 3.3 mm long) with mottled brown wings. The adults are 

active and can readily fly over short distances when disturbed. Adults may be 

found resting or feeding on leaves with their bodies held at a 45° angle to the leaf 

surface (Hall, 2008). The psyllid is highly prolific in the presence of new shoots 

and under favorable environmental conditions (average daily temperature 

between 24 and 29°C and relative humidity between 50 and 75%) (Aubert, 1987; 

Tsai and Liu, 2000; Nava et al., 2007). Epidemiological and spatial distribution 

studies have shown that the psyllid is usually dispersed between trees over 

distances of 25 to 50m, reaching up to 200m (Gottwald et al., 2007; Boina et al., 

2009; Tomaseto et al., 2016). 

The population dynamics of ACP is consistently associated with the 

presence of new shoots in citrus trees and favorable environmental conditions for 

their development (Husain and Nath, 1927; Tsai and Liu, 2000; Hall et al., 2008). 

Higher populations generally occur in spring and summer, while lower 

populations occur in autumn and winter, when the trees are in vegetative rest and 

new shoot growth is reduced (Yamamoto et al., 2001).  

 



11 
 

2.6 Rootstock responses to HLB 

 

Although all citrus trees are susceptible to HLB, different hosts have shown 

distinct responses to Las infection. The tolerance to HLB is a poorly understood 

phenomenon. Trifoliate orange and some of its hybrids have been classified as 

tolerant to Las infection, due to low bacterial titers and less severe symptoms on 

the leaves, as observed on potted seedlings of ‘Carrizo’ citrange (C. × aurantium 

var. sinensis L. × P. trifoliata) compared to ‘Volkamer’ lemon and ‘Cleopatra’ 

mandarin (Folimonova et al., 2009). Lower percentages of Las-positive trees 

were found in potted seedlings of ‘Carrizo’ citrange than ‘Cleopatra’ mandarin or 

‘Volkamer’ lemon, and less severe leaf symptoms were observed on seedlings of 

the trifoliate hybrid US-897 (‘Cleopatra’ × P. trifoliata ‘Flying Dragon’) than on 

‘Cleopatra’ mandarin (Albrecht and Bowman, 2012). 

In addition to leaf symptoms and bacterial titer, the capacity of the 

rootstock to replace roots damaged by Las colonization was associated with HLB-

tolerance (Wu et al., 2017; 2018). In Florida, root losses of up to ~ 37% were 

detected in HLB-symptomatic trees of 'Hamlin' and 'Valencia' sweet oranges 

grafted on 'Swingle' citrumelo compared to asymptomatic trees (Graham et al., 

2013). Root damage takes place even before symptoms are detected on the 

scion. HLB-symptomatic trees of 'Valencia Late' grafted on 'Swingle 'citrumelo 

presented ca. 26% less root dry weight than asymptomatic trees (Johnson et al., 

2014). 

In the field, yield has been used as an important attribute to classify the 

tolerance to HLB. Under natural inoculation condition, five hybrids originated from 

a cross between ‘Changsha’, ‘Cleopatra’ or ‘Ninnkat’ mandarins and ‘Gotha 

Road’ trifoliate induced higher yield of ‘Hamlin’ sweet orange than ‘Swingle’ 

citrumelo or ‘Flying Dragon’ trifoliate (Bowman and McCollum, 2015). Another 

field trial demonstrated that yield was higher on ‘Valencia Late’ grafted on US-

942 (‘Sunki’ x P. trifoliata ‘Flying Dragon’) than on ‘Cleopatra’ mandarin and 

‘Carrizo’ citrange (Bowman et al., 2016).  

 

 

 



12 
 

2.7 Translocation of Candidatus Liberibacter spp. 

 

Carbohydrates are mainly transported in the phloem of vascular plants in 

the form of sucrose. The photoassimilates produced by photosynthesis or the 

hydrolyzed sugars from reserve compounds are distributed and used by the plant 

to grow and generate energy during the cellular respiration process (Taiz et al., 

2017). 

In phloem, sap is transported from production areas, called sources, to 

areas of active growth or storage, called sinks. The sources are usually mature 

leaves, which are able to produce photoassimilates, which export them during a 

given stage of their development. Sinks include non-photosynthetic plant organs 

and organs that do not produce enough photoassimilates, such as roots, 

developing fruits and immature leaves (Taiz et al., 2017). 

Many pathogens colonize the phloem, like viruses and phytoplasmas. 

These pathogens that do not have active transport structures are probably 

translocated within the plants following the phloem sap flow from the source to 

the sink regions (Leisner and Turgeon, 1993; Roberts et al., 1997). Active 

transport structures such as flagella and pili were not observed in microscopic 

images of Las (Andrade et al., 2019; Hilf et al., 2013), suggesting it moves 

passively in the phloem, similar to viruses and phytoplasmas (Christensen et al., 

2004; Dawson et al., 2013; Folimonova et al., 2008; Jiang et al., 2004). 

Apparently, Las movement within citrus trees follows the source-sink 

translocation pattern of phloem sap. Recently, Las cells were detected on the 

plasma membrane of the plant cells next to the phloem pores, suggesting the 

pores may be involved in the cell-to-cell movement of the bacterium (Achor et al., 

2020). It has also been shown that Las is widely distributed, reaching several 

parts of the tree (Tatineni et al., 2008; Li et al., 2009). In citrus, bacterial 

translocation follows the phloem sap flow, first colonizing young tissues that are 

serving as sinks (Raiol-Junior et al., 2020; Hilf and Luo, 2018). This behavior is 

similar to that found for the ‘Ca. L. solanacearum’, associated with the zebra chip 

disease in potatoes (Solanum tuberosum) and tomatoes (Solanum 

lycopersicum), in which the pathogen also was first detected in young tissues 

after inoculation (Levy et al., 2011). 



13 
 

The rootstock may play an important role in the process of colonization 

and multiplication of the pathogen. It has been shown that losses in the roots 

occur even before symptoms are seen on the scion (Raiol-Junior et al., 2020; 

Johnson et al., 2014). In citrus, the rootstock vigor may influence the growth and 

development of young tissues and the flushing pattern on the scion. As a result, 

depending on the vigor, the rootstock can induce a higher or lower translocation 

of photoassimilates, which may, indirectly, influence the translocation of Las in 

the tree. Consequently, Las colonization may be influenced by the scion and 

rootstock combination used. Finding a rootstock that delays Las colonization 

within citrus trees can contribute to the longevity of trees in orchards and reduce 

losses caused by the disease. 

 

3. REFERENCES 

 

Achor D, Welker S, Ben-Mahmoud S, Wang C, Folimonova SY, Dutt M, Gowda 
S, Levy A (2020) Dynamics of Candidatus Liberibacter asiaticus Movement and 
Sieve-Pore Plugging in Citrus Sink Cells. Plant Physiology 182:882-891. 

ADAPAR 2019: agência de defesa agropecuária do Paraná (2019) Coordenação 
do Programa da Sanidade da Citricultura. Relatório Anual, Curitiba. Available at 
:< http://www.adapar.pr.gov.br/>. 

Agostini JP, Preussler C, Outi Y (2015) Huanglongbing (HLB) ex greening. In.: 
Rossini MN, Dummel DM, Agostini JP (Eds.) Plagas cuarentenarias de frutales 
de la República Argentina: avances en los resultados. INTA, Argentina, p. 
183-198. 

AGRIANUAL 2020: anuário da agricultura brasileira. São Paulo: FNP Consultoria 
& Comércio. 416 p. 

Ahn SJ, Im YJ, Chung GC, Cho BH, Suh SR (1999) Physiological response of 
grafted cucumber leaves and rootstock roots affected by low root temperature. 
Scientia Horticulturae 81:397-408. 

Albrecht U, Bowman KD (2012) Tolerance of trifoliate citrus rootstock hybrids to 
Candidatus Liberibacter asiaticus. Scientia Horticulturae 147:71-80. 

Albrecht U, McCollum G, Bowman KD (2012) Influence of rootstock variety on 
huanglongbing disease development in field-grown sweet orange (Citrus sinensis 
[L.] Osbeck) trees. Scientia Horticulturae 138:210-220. 

Andrade MO, Pang Z, Achor DS, Wang H, Yao T, Singer BH, Wang N (2019) The 
flagella of ‘Candidatus Liberibacter asiaticus’ and its movement in planta. 
Molecular Plant Pathology 21:109-123. 

http://www.adapar.pr.gov.br/


14 
 

Atkinson CJ, Else MA, Taylor L (2003) Root and stem hydraulic conductivity as 
determinants of growth potential in grafted trees of apple (Malus pumila Mill.). 
Journal of Experimental Botany 54:1221-1229.  

Aubert B (1987) Trioza erytreae Del Guercio and Diaphorina citri Kuwayama 
(Homoptera: Psyllidae), the two vectors of citrus greening disease: biological 
aspects and posible control strategies. Fruits 42:149-162. 

Barry GH, Caruso M, Gmitter JR FG (2020) Commercial scion varieties. In.: Talon 
M, Caruso M, Gmitter Jr FG (Eds.) The Genus Citrus. Amsterdam: Elsevier, p. 
83-104. 

Bassanezi RB, Lopes SA, Belasque Jr J, Spósito MB, Yamamoto PT, Miranda 
MP, Teixeira DC, Wulff NA (2010) Epidemiologia do huanglongbing e suas 
implicações para o manejo da doença. Revista Citrus Research & Technology 
31:11-23. 

Bassanezi RB, Lopes SA, Miranda MP, Wulff NA, Volpe HXL, Ayres AJ (2020) 
Overview of citrus huanglongbing spread and management strategies in Brazil. 
Tropical Plant Pathology 45:251-264. 

Belasque Jr J, Yamamoto PT, Miranda MP, Bassanezi RB, Ayres AJ, Bové JM 
(2010) Controle do huanglongbing no estado de São Paulo, Brasil. Revista 
Citrus Research & Technology 31: 53-64. 

Blumer S, Pompeu Junior J (2005) Avaliação de citrandarins e outros híbridos de 
trifoliata como porta-enxertos para citros em São Paulo. Revista Brasileira de 
Fruticultura 27:264-267. 

Boava LP, Cristofani-Yaly M, Machado MA (2017) Physiologic, Anatomic, and 
Gene Expression Changes in Citrus sunki, Poncirus trifoliata, and Their Hybrids 
After ‘Candidatus Liberibacter asiaticus’ Infection. Phytopathology 107:590-
599. 

Boina DR, Meyer WL, Onagbola EO, Stelinski LL (2009) Quantifying dispersal of 
Diaphorina citri (Hemiptera: Psyllidae) by immunomarking and potential impact 
of unmanaged groves on commercial citrus management. Environmental 
Entomology 38:1250-1258. 

Bové JM (2006) Huanglongbing: A destructive, newly-emerging, century-old 
disease of citrus. Journal of Plant Pathology 88:7-37. 

Bowman KD, Joubert J (2020) Citrus rootstocks. In.: Talon M, Caruso M, Gmitter 
Jr FG (Eds.) The Genus Citrus. Amsterdam: Elsevier, p. 105-127. 

Bowman KD, McCollum G (2015) Five new citrus rootstocks with improved 
tolerance to huanglongbing. HortScience 50:1731-1734. 

Bowman KD, McCollum G, Albrecht U (2016) Performance of 'Valencia' Orange 
(Citrus sinensis [L.] Osbeck) on 17 rootstocks in a trial severely affected by 
huanglongbing. Scientia Horticulturae 201:355-361. 



15 
 

Capoor S, Rao D, Viswanath S (1967) Diaphorina citri Kuwayama, a vector of the 
greening disease of citrus in India. Indian Journal of Agricultural Science 
37:572-579. 

Carvalho SAD, Girardi EA, Mourão Filho FAA, Ferrarezi RS, Coletta Filho HD 
(2019) Advances in citrus propagation in Brazil. Revista Brasileira de 
Fruticultura 41: e-422.  

Castro MEA, Bezerra AR, Leite WA, Mundin Filho W, Nogueira ND (2010) 
Situação e ações do estado de Minas Gerais frente ao huanglongbing. Citrus 
Research Technology 31:163-168. 

Chapot H (1975) The Citrus plant. Switzerland: Basle Ciba-Geigy 
Agrochemicals, p. 6-13. 

Christensen NM, Nicolaisen M, Hansen M, Schulz A (2004) Distribution of 
phytoplasmas in infected plants as revealed by real-time PCR and bioimaging. 
Molecular Plant-Microbe Interactions 17:1175-1184. 

Cifuentes-Arenas JC, Goes A, Miranda MP, Beattie GAC, Lopes SA (2018) Citrus 
flush shoot ontogeny modulates biotic potential of Diaphorina citri. PloS One 13: 
e0190563. 

CITRUSBR 2020. Geração de empregos na citricultura cresceu em 2019. 
Available on: < http://www.citrusbr.com/mercado/>. Accessed on: May. 24th, 
2020. 

Coletta-Filho HD, Targon MLPN, Takita MA, De Negri JD, Pompeu Jr J, Machado 
MA, Amaral AM, Muller GW (2004) First Report of the Causal Agent of 
Huanglongbing (“Candidatus Liberibacter asiaticus”) in Brazil. Plant Disease 
88:1382-1382. 

Costa Lima AM (1942) Insetos do Brasil: Homopteros. Rio de Janeiro: Escola 
Nacional de Agronomia, 327 p. 

Da Graça JV (1991) Citrus Greening Disease. Annual Review Phytopathology 
29:109-136. 

Davies FS, Albrigo LG ( 1994) Citrus. Wallingford: Cab International, 254 p. 

Dawson WO, Garnsey SM, Tatineni S, Folimonova SY, Harper SJ, Gowda S 
(2013) Citrus tristeza virus-host interactions. Frontiers in Microbiology 4:1-10. 

De Carli LF, Miranda MP, Volpe HXL, Zanardi OZ, Vizoni MC, Martini FM, Lopes 
JAP (2018) Leaf age affects the efficacy of insecticides to control Asian citrus 
psyllid, Diaphorina citri (Hemiptera: Liviidae). Journal of Applied Entomology 
142:689-695. 

Diário Oficial da União (2019) Instrução Normativa N° 26, de 10 de setembro de 
2019, do Ministério da Agricultura, Pecuária e Abastecimento. Available at:< 
https://www.in.gov.br/web/dou/-/instrucao-normativa-n-26-de-10-de-setembro-
de-2019-216065360> Accessed on: May. 24th, 2020. 

http://www.citrusbr.com/mercado/
https://www.in.gov.br/web/dou/-/instrucao-normativa-n-26-de-10-de-setembro-de-2019-216065360
https://www.in.gov.br/web/dou/-/instrucao-normativa-n-26-de-10-de-setembro-de-2019-216065360


16 
 

EPPO 2016: european and mediterranean plant protection organization. 
Available at: <https://gd.eppo.int/taxon/DIAACI/distribution/>;< 
https://gd.eppo.int/taxon/TRIZER/distribution/ >. Accessed on: May 24, 2020.  

FAO 2020: Food and agriculture organization of the united nations. FAOSTAT. 
Available at: < http://www.fao.org/faostat/en/ >. Accessed on: May 24, 2020.  

Figueiredo JO (1991) Variedades copas. Citricultura brasileira. Campinas: 
Fundação Cargill 228-57. 

Folimonova SY, Folimonov AS, Tatineni S, Dawson WO (2008) Citrus tristeza 
virus: survival at the edge of the movement continuum. Journal of Virology 
82:6546-6556. 

Folimonova SY, Robertson CJ, Garnsey CS, Gowda S, Dawson WO (2009) 
Examination of the responses of different genotypes of citrus to huanglongbing 
(citrus greening) under different conditions. Phytopathology 99:1346-1354. 

Forner-Giner MA, Rodriguez-Gamir J, Martinez-Alcantara B, Quiñones A, 
Iglesias DJ, Primo-Millo E, Forner J (2014) Performance of Navel orange trees 
grafted onto two new dwarfing rootstocks (Forner-Alcaide 517 and Forner-
Alcaide 418). Scientia Horticulturae 179:376-387. 

Fundecitrus 2020a: Fundo de Defesa da Citricultura. Tree inventory and 2020-
2021 orange crop forecast for the São Paulo and west-southwest Minas Gerais 
citrus belt. Araraquara, SP, 141 p. Available on: < 
https://www.fundecitrus.com.br/pdf/pes_relatorios/2020_06_25_Tree_Inventory
_and_Orange_Crop_Forecast_2020-2021.pdf>. Accessed on: Aug. 9th, 2020. 

Fundecitrus 2020b: Fundo de Defesa da Citricultura. Levantamento da incidência 
das doenças dos citros: Greening, CVC e cancro cítrico no do cinturão citrícola 
de São Paulo e Triângulo/Sudoeste Mineiro. Araraquara, SP, 67 p. Available at: 
< https://www.fundecitrus.com.br/pdf/levantamentos/levantamento-doencas-
2020.pdf >. Accessed on: Aug. 9th, 2020. 

Garieri DS (2016) Estudo da brotação de laranjeira Natal sobre sete 
diferentes porta-enxertos visando o controle de Diaphorina citri. 28 f. 
Dissertação (Mestrado Profissional em Controle de Doenças e Pragas dos 
Citrus) – Fundecitrus, Araraquara. 

Garnier M, Danel N, Bové JM (1984) The greening organism is a Gram negative 
bacterium. In.: Proceedings of 9th Conference of International Organization 
of Citrus Virologists. Riverside, CA, p. 115-124. 

Gasparoto MCG, Coletta-Filho HD, Bassanezi RB, Lopes SA, Lourenço SA, 
Amorim L (2012) Influence of temperature on infection and establishment of 
‘Candidatus Liberibacter americanus’ and ‘Candidatus Liberibacter asiaticus’ in 
citrus plants. Plant Pathology 61:658-664. 

Gimenes-Fernandes N, Bassanezi RB (2001) Doença de causa desconhecida 
afeta pomares cítricos no norte de São Paulo e sul do Triângulo Mineiro. Summa 
Phytopathologica 27:93. 

http://www.eppo.int/
https://gd.eppo.int/taxon/DIAACI/distribution/
https://gd.eppo.int/taxon/TRIZER/distribution/
http://www.fao.org/faostat/en/
https://www.fundecitrus.com.br/pdf/pes_relatorios/2020_06_25_Tree_Inventory_and_Orange_Crop_Forecast_2020-2021.pdf
https://www.fundecitrus.com.br/pdf/pes_relatorios/2020_06_25_Tree_Inventory_and_Orange_Crop_Forecast_2020-2021.pdf
https://www.fundecitrus.com.br/pdf/levantamentos/levantamento-doencas-2020.pdf
https://www.fundecitrus.com.br/pdf/levantamentos/levantamento-doencas-2020.pdf


17 
 

Gottwald TR, Da Graça JV, Bassanezi RB (2007) Citrus Huanglongbing: the 
pathogen and its impact. Plant Health Progress. Doi: 10.1094/PHP2007-0906-
01 RV. 

Grafton-Cardwell EE, Stelinski LL, Stansly PA (2013) Biology and management 
of asian citrus psyllid, vector of the huanglongbing pathogens. Annual Review 
Entomology 58:413-432. 

Graham JH, Johnson EG, Gottwald TR, Irey MR (2013) Presymptomatic fibrous 
root decline in citrus trees caused by huanglongbing and potential interaction with 
Phytophthora spp. Plant Disease 97:1195-1199. 

Halbert SE (2005) The discovery of huanglongbing in Florida. In.: Proceedings 
of 2nd international citrus canker and huanglongbing research workshop. 
Orlando, FL, p. 50. 

Hall DG (2008) Biology, history and world status of Diaphorina citri. In.: 
Proceedings of the international workshop on huanglongbing and Asian 
citrus psyllid. Orlando, FL, p. 1-11. 

Hall DG, Hentz MG, Adair JR RC (2008) Population ecology and phenology of 
Diaphorina citri (Hemiptera: Psyllidae) in two Florida citrus groves. 
Environmental Entomology 37:914-924. 

Hall DG, Albrecht U, Bowman KD (2016) Transmission rates of ‘Ca. Liberibacter 
asiaticus’ by Asian citrus psyllid are enhanced by the presence and 
developmental stage of citrus flush. Journal of Economic Entomology 
109:558-563. 

Hall DG, Richardson ML, Ammar ED, Halbert SE (2013) Asian citrus psyllid, 
Diaphorina citri, vector of citrus huanglongbing disease. Entomologia 
Experimentalis et Applicata 146:207-223. 

Hasse G (1987) A laranja no Brasil 1500-1987. São Paulo: Edição de Duprat; 
Iobe Propaganda, 296 p. 

Hilf ME, Luo W (2018) Dynamics of ‘Candidatus Liberibacter asiaticus’ 
colonization of new growth of citrus. Phytopathology 108:1165-1171. 

Hilf ME, Sims KR, Folimonova SY, Achor DS (2013) Visualization of ‘Candidatus 
Liberibacter asiaticus’ cells in the vascular bundle of citrus seed coats with 
fluorescence in situ hybridization and transmission electron microscopy. 
Phytopathology 103:545-554. 

Husain MA, Nath D (1927) The citrus psylla (Diaphorina citri, Kuw.) (Psyllidae: 
Homoptera). Memoirs of the Department of Agriculture in India 10:1–27. 

Hutchison DJ (1974) Swingle citrumelo-a promising rootstock hybrid. In.: 
Proceedings of the Florida State Horticultural Society, Lake Alfred, FL, p. 89-
91. 



18 
 

IBGE 2020: Instituto Brasileiro de Geografia e Estatística. Available at: < 
https://sidra.ibge.gov.br/home/pms/brasil >. Accessed on: Aug 9th, 2020.  

ICA 2019: Instituto Colombiano Agropecuário. Sistema de Alerta Fitossanitário, 
2015. Resolución No2390. Available at: 
<:http://extwprlegs1.fao.org/docs/pdf/col151548.pdf>. Accessed on: Sept. 21, 
2019. 

Instituto Mineiro De Agropecuária / Gerência De Defesa Sanitária Vegetal (2019) 
Delimitação de áreas de risco para adoção de medidas de prevenção e 
erradicação do HLB (Greening) em Minas Gerais de acordo com a portaria IMA 
no. 1,649 de 18 de agosto de 2016 (Atualização: 02/09/2019). Available at: 
http://ima. mg.gov.br/material-curso-cfo-cfoc/doc_details/3925-area-de-riscode-
greening-setembro-2019. Accessed on: Sept. 28, 2019. 

Jagoueix S, Bove JM, Garnier M (1994) The phloem-limited bacterium of 
greening disease of citrus is a member of the alpha subdivision of the 
Proteobacteria. International Journal of Systematic Bacteriology 44:397-486. 

Jiang H, Wei W, Saiki T, Kawakita H, Watanabe K, Sato M (2004) Distribution 
patterns of mulberry dwarf phytoplasma in reproductive organs, winter buds, and 
roots of mulberry trees. Journal of General Plant Pathology 70:168-173. 

Johnson EG, WU J, Bright DB, Graham JH (2014) Association of “Candidatus 
Liberibacter asiaticus” root infection, but not phloem plugging with root loss on 
huanglongbing-affected trees prior to appearance of foliar symptoms. Plant 
Pathology 63:290-298. 

Kistner EJ, Amrich R, Castillo M, Strode V, Hoddle MS (2016) Phenology of Asian 
citrus psyllid (Hemiptera: Liviidae), with special reference to biological control by 
Tamarixia radiata, in the residential landscape of southern California. Journal of 
Economic Entomology 109:1047-1057. 

Leisner SM, Turgeon R (1993) Movement of virus and photoassimilate in the 
phloem: A comparative analysis. Bioessays 15:741-748. 

Levy J, Ravindran A, Gross D, Tamborindeguy C, Pierson E (2011) Translocation 
of ‘Candidatus Liberibacter solanacearum’, the zebra chip pathogen, in potato 
and tomato. Phytopathology 101:285-1291. 

Li W, Levy L, Hartung JS (2009) Quantitative distribution of “Candidatus 
Liberibacter asiaticus” in citrus plants with citrus huanglongbing. 
Phytopathology 99:139-144. 

Lopes SA, Bertolini E, Frare GF, Martins EC, Wulff NA, Teixeira DC, Fernandes, 
NG, Cambra M (2009b) Graft transmission efficiencies and multiplication of 
Candidatus Liberibacter americanus and Ca. Liberibacter asiaticus in citrus 
plants. Phytopathology 99:301-306. 

Lopes SA, Frare GF, Bertolini E, Cambra M, Fernandes NG, Ayres AJ, Marin DR, 
Bové JM (2009a) Liberibacters associated with citrus huanglongbing in Brazil: 

https://sidra.ibge.gov.br/home/pms/brasil
http://extwprlegs1.fao.org/docs/pdf/col151548.pdf


19 
 

Candidatus Liberibacter asiaticus is heat tolerant, Ca. L. americanus is heat 
sensitive. Plant Disease 93:257-262. 

Lopes SA, Luiz FQBF, Martins EC, Fassini CG, Sousa MC, Barbosa JC, Beattie 
GAC (2013) ‘Candidatus Liberibacter asiaticus’ titers in citrus and acquisition 
rates by Diaphorina citri are decreased by higher temperature. Plant Disease 
97:1563-1570. 

Lopes SA, Luiz FQBF, Oliveira HT, Cifuentes-Arenas JC, Raiol-Junior LL (2017) 
Seasonal variation of ‘Candidatus Liberibacter asiaticus’ titers in new shoots of 
citrus in distinct environments. Plant Disease 101:583–590. 

Luis M, Collazo C, et al. (2009) Occurrence of citrus Huanglongbing in Cuba and 
association of the disease with Candidatus Liberibacter asiaticus. Journal Plant 
Pathology 91:709-712. 

Matos L, Hilf ME, Camejo J (2009) First report of “Candidatus Liberibacter 
asiaticus” associated with citrus huanglongbing in the Dominican Republic. Plant 
Disease 93:668-668. 

Mattos Junior D, Negri JD, Pio RM, Pompeu Junior J (2005) Citros. Campinas: 
IAC, 929 p. 

McClean APD, Oberholzer PCJ (1965) Citrus psyllid, a vector of the greening 
disease of sweet orange. South African Journal of Agricultural Science 8:297-
298. 

Merwe AJ, Andersen FG (1937) Chromium and manganese toxicity: Is it 
important in Transvaal citrus greening? Farming South Africa 12:439-440. 

Molina-Bravo R, Stephens-Cárdenas SA, Hilje-Rodríguez I, Blanco-Vargas M, 
Villalobos-Navarro D, Gätjens-Boniche O, Moreira-Carmona L, Villalobos-Müller 
W (2015) Citrus huanglongbing associated with ‘Candidatus liberibacter 
asiaticus’ present in the northern region of Costa Rica but has not been detected 
in other citrus-growing areas. Plant Disease 99:1855-1855. 

Nava DE, Torres MLG, Rodrigues MDL, Bento JMS, Parra JRP (2007) Biology 
of Diaphorina citri (Hem., Psyllidae) on different hosts and at different 
temperatures. Journal of Applied Entomology 131:709-715. 

Nunes WMC, Souza EB, Leite Junior RP, Salvador CA, Rinaldi DA, Croce Filho 
J, Paiva PG (2010) Plano de ação para o controle de huanglongbing no estado 
do Paraná, Brasil. Citrus Research Technology 31:169–177. 

Oliveira HT (2017) Dinâmica de brotação em diferentes combinações de 
copa e porta-enxerto de citros em área irrigada e não irrigada. 37 f. 
Dissertação (Mestrado Profissional em Controle de Doenças e Pragas dos 
Citrus) - Fundecitrus, Araraquara-SP. 

Olmstead MA, Lang NS, Ewers FW, Owens SA (2006) Xylem vessel anatomy of 
sweet cherries grafted onto dwarfing and nondwarfing and nondwarfing 



20 
 

rootstocks. Journal of the American Society for Horticultural Science 
131:577-85. 

Pio RM, Figueiredo JO, Stuchi ES, Cardoso SAB (2005) Variedades copas. In.: 
Mattos Junior D, Negri JD, Pio RM, Pompeu Junior J (Eds.) Citros. Campinas: 
IAC, p. 37-60. 

Pompeu Junior J (2001) Rootstocks and scions in the citriculture of the São Paulo 
State. In.: Proceedings of International Congress of Citrus Nurserymen. 
Ribeirão Preto, BR, 6:75-82. 

Pompeu Junior J (2005) Porta-enxertos. In.: Mattos Junior D, Negri JD, Pio RM, 
Pompeu Junior J (Eds.) Citros. Campinas: IAC, p. 61-104. 

Pompeu Junior J, Blumer S (2014) Híbridos de trifoliata como porta-enxertos 
para laranjeira ‘Pera’. Pesquisa Agropecuária Tropical 1: 9-14. 

Pompeu Junior J, Blumer S, Pompeu GB (2008) Tangerineiras como porta-
enxertos para laranjeira 'Pera'. Ciência e Agrotecnologia 32: 1218-1223. 

Pompeu Junior J, Laranjeira FF, Blumer S (2002) Laranjeiras 'Valência' 
enxertadas em híbridos de trifoliata. Scientia Agricola 59:93-97. 

Primo-Millo E, Agustí M (2020) Flowering and fruit set. In.: Talon M, Caruso M, 
Gmitter Jr FG (Eds.) The Genus Citrus. Amsterdam: Elsevier, p. 219-244. 

Raiol-Junior LL, Cifuentes-Arenas JC, Carvalho EV, Girardi EA, Lopes SA (2020) 
Evidence that ‘Candidatus Liberibacter asiaticus’ moves predominantly towards 
new tissue growth in citrus plants. Plant disease. Doi: 10.1094/PDIS-01-20-
0158-RE. 

Ramos YC, Stuchi ES, Girardi EA, Leão HC, Gesteira AS, Passos OS, Soares 
Filho WS (2015) Dwarfing rootstocks for 'Valencia' sweet orange. Acta 
Horticulturae 1065:351-354. 

Reinking OA (1919) Diseases of economic plants in southern China. Philippine 
Agriculturist 8:109-135. 

Roberts AG, Santa Cruz S, Roberts IM, Prior DAM, Turgeon R, Oparka KJ (1997) 
Phloem unloading in sink leaves of Nicotiana benthamiana comparison of a 
fluorescent solute with a fluorescent virus. Plant Cells 9:1381-1396. 

Rodrigues JDB (2018) Desempenho horticultural e tolerância ao 
huanglongbing de combinações de laranjeira ‘Valência’ enxertada em 
diferentes porta-enxertos. 83 f. Dissertação (Mestrado em Agronomia - 
Genética e Melhoramento de Plantas) - Unesp, Jaboticabal-SP. 

Rodriguez O, Rossetti V, Müller GW  (1979) Declínio de plantas cítricas em 
São Paulo. In.: Congresso Brasileiro de Fruticultura. Pelotas, RS, p. 927-932. 

Scora RW (1975) On the history and origin of Citrus. Bulletin of the Torrey 
Botanical Club 102:369-375. 

https://doi.org/10.1094/PDIS-01-20-0158-RE
https://doi.org/10.1094/PDIS-01-20-0158-RE


21 
 

Senasave 2019: Servicio Nacional de Calidad y Sanidad Vegetal y de Semillas, 
2013. Resolución N° 80 “Por la cual se declara emergencia fitosanitaria por la 
presencia de la plaga denominada Huanglongbing (HLB) de los cítricos, en todo 
el territorio nacional” Available at: 
<http://web.senave.gov.py:8081/docs/web/hlb/Informe%20Diagnostico%20Epi 
emiologico%20Regional%20HLB_Paraguay_03Mar14.pdf>. Accessed on: Sept. 
21, 2019. 

Sétamou M, Alabi OJ, Kunta M, Jifon JL, Da Graça JV (2016) Enhanced 
acquisition rates of ‘Candidatus Liberibacter asiaticus’ by the Asian citrus psyllid 
(Hemiptera: Liviidae) in the presence of vegetative flush growth in citrus. Journal 
of Economic Entomology 109:1973-1978. 

Shimwela MM, Narouei-Khandan HA, Halbert SE, Keremane ML, Minsavage GV, 
Timilsina S, Van Bruggen AH (2016) First occurrence of Diaphorina citri in East 
Africa, characterization of the Candidatus Liberibacter species causing 
huanglongbing (HLB) in Tanzania, and potential further spread of D. citri and HLB 
in Africa and Europe. European Journal of Plant Pathology 146:1-20. 

Soumelidou K, Morris DA, Battey NH, Barnett JR, John P (1994) Auxin transport 
capacity in relation to the dwarfing effect of apple rootstocks. Journal of 
Horticultural Science 69:719-25. 

Spiegel-Roy P, Goldschmidt EE (1996) Biology of citrus. Cambridge: 
Cambridge University Press, 230 p. 

Stuchi ES, Girardi EA, Sempionato OR, Reiff ET, Silva SR, Parolin LG (2012) 
Trifoliata ‘Flying Dragon’: Porta-enxerto para plantios adensados e 
irrigados de laranjeiras doces e alta produtividade e sustentabilidade. Cruz 
das Almas: Embrapa Mandioca e Fruticultura, 7 p. 

Swingle WT, Reece PC (1967) The botany of citrus and its wild relatives. In.: 
Reuther W, Webber HJ, Batchelor LD (Eds.) The citrus industry. Riverside: 
University of California, 1:190-430. 

Taiz L, Zeiger E, Møller IM, Murphy A (2017) Plant physiology and 
development. Porto Alegre: Artmed, 858 p. 

Tatineni S, Sagaram US, Gowda S, Robertson CJ, Dawson WO, Iwanami T, 
Wang N (2008) In Planta Distribution of “Candidatus Liberibacter asiaticus” as 
Revealed by Polymerase Chain Reaction (PCR) and Real-Time PCR. 
Phytopathology 98:592-599. 

Teck SLC, Fatimah A, Beattie GAC, Heng RKJ, King WS. (2011). Seasonal 
population dynamics of the Asian citrus psyllid, Diaphorina citri Kuwayama in 
Sarawak. American Journal of Agricultural and Biological Sciences 64:527-
535. 

Teixeira DC, Saillard C, Eveillard S, Danet JL, Da Costa PI, Ayres AJ, Bové JM 
(2005) Candidatus Liberibacter americanus, associated with citrus 
huanglongbing (greening disease) in São Paulo State, Brazil. International 
Journal of Systematic and Evolutionary Microbiology 55:1857-1862. 

http://web.senave.gov.py:8081/docs/web/hlb/Informe%20Diagnostico%20Epi%20emiologico%20Regional%20HLB_Paraguay_03Mar14.pdf
http://web.senave.gov.py:8081/docs/web/hlb/Informe%20Diagnostico%20Epi%20emiologico%20Regional%20HLB_Paraguay_03Mar14.pdf


22 
 

Tomaseto AF, Krugner R, Lopes JRS (2016) Effect of plant barriers and citrus 
leaf age on dispersal of Diaphorina citri (Hemiptera: Liviidae). Journal of Applied 
Entomology 140:91-102. 

Tsai JH, Liu YH (2000) Biology of Diaphorina citri (Hemiptera: Psyllidae) on four 
host plants. Journal of Economic Entomology 93:1721-1725. 

Vidalakis G, Pagliaccia D, Bash JA, Afunian M, Semancik JS (2011) Citrus 
dwarfing viroid: effects on tree size and scion performance specific to Poncirus 
trifoliata rootstock for high-density planting. Annals of Applied Biology 158:204-
217. 

Wu GA, Terol J, et al. (2018) Genomics of the origin and evolution of Citrus. 
Nature 554:311-316. 

Wu J, Johnson EG, Bright DB, Gerberich, KM, Graham JH (2017) Interaction 
between Phytophthora nicotianae and Candidatus Liberibacter asiaticus damage 
to citrus fibrous roots. Journal of Citrus Pathology 1-7. 

Wu J, Johnson EG, Gerberich KM, Bright DB, Graham JH (2018) Contrasting 
canopy and fibrous root damage on Swingle citrumelo caused by ‘Candidatus 
Liberibacter asiaticus’ and Phytophthora nicotianae. Plant Pathology 67:202-
209. 

Yamamoto PT, Felippe MR, Garbim LF, Coelho JHC, Ximenes NL, Martins EC 
(2006) Diaphorina citri (Kuwayama) (Hemiptera: Psyllidae): vector of the 
bacterium Candidatus Liberibacter americanus. In.: Proceedings of the 
Huanglongbing - Greening International Workshop, Ribeirão Preto, SP, p. 96. 

Yamamoto PT, Paiva PE, Gravena S (2001) Population dynamics of Diaphorina 
citri Kuwayama (Hemiptera: Psyllidae) in citrus orchards in the North of São Paulo 
State, Brazil. Neotropical Entomology 30:165-170. 

 
 

 

 

 

 

 

 

 

 



23 
 

CHAPTER 2 - Modeling seasonal flushing and shoot growth on different 

citrus scion-rootstocks  

 

 

ABSTRACT – Occurrence, intensity, and growth patterns of new shoots 
(NS) were evaluated on different scion and rootstock combinations in a 2.5-year-
old citrus orchard in Bebedouro, SP, Brazil, with the aim of improving timing of 
insecticide applications to suppress incidence of Diaphorina citri in citrus 
orchards. ‘Pera’ and ‘Folha Murcha’ sweet oranges, ‘Ponkan’ mandarin and 
‘Persian’ lime were growing on a set of nine hybrid rootstocks including 
commercial rootstocks on which growth of scions differ. NS were counted every 
20 days from August 2017 to December 2018 within a square frame (0.25 m2) 
set on the central portion of the canopy of eight trees per scion-rootstock 
combination, and individually classified in six vegetative stages of development. 
The number of NS and the area under flush shoot dynamics (AUFSD) were used 
to compare NS intensities. Mean NS number (dependent variable) and maximum, 
minimum and average temperatures (°C) and accumulated rainfall (mm) at 20, 
40 and 60 days prior to the evaluation date (independent variables) were used to 
develop multiple linear regression models to describe NS occurrence dynamics. 
Individual NS growth was evaluated during the spring of 2018 and autumn of 
2019 and the data compared with the output of Logistic or Gompertz non-linear 
regression models based on degree days (DD). NS occurrence dynamics were 
similar for all combinations, but the intensity was significantly higher on vigorous 
‘Rough’ lemon rootstock than on dwarfing ‘Flying Dragon’ trifoliate rootstock. 
Occurrence of NS on ‘Pera’, ‘Folha Murcha’ and ‘Persian’ lime associated 
positively and significantly (P < 0.05) with an increase of minimum and average 
temperatures (avg. adjusted R2 = 0.406; 0.408; 0.403, respectively) and 
occurrence of NS on ‘Ponkan’ with accumulated rainfall (avg. adj. R2 = 0.311), 
registered during 20- or 40-day intervals prior the evaluation date. NS grew faster 
and attained longer lengths during spring (34 days and 102.7 mm) than autumn 
(42 days and 71.9 mm), except for ‘Folha Murcha’ which took longer to grow and 
reached shorter lengths (38 days and 78.8 mm) during spring, and longer lengths 
during autumn (42 days and 118.3 mm). Logistic models better fitted NS growth 
data (avg. adj. R2 ≥ 0.94). The adjusted R2 values of regression models 
developed to predict NS occurrences during the study were not high enough to 
estimate optimum timing to start insecticide applications for control of D. citri. 
However, since NS growth consists of foliar tissues that initially are highly suitable 
for psyllid feeding and oviposition, the adjusted R2 values attained for NS growth 
can be used to improve timing, and thereby adjust frequency of insecticide 
applications.   

 

Keywords: Citrus spp., Poncirus trifoliata, phenological stages, plant growth, 
meteorological conditions, modeling, non-linear regression 
 

 



24 
 

1. INTRODUCTION 

 

Vegetative growth of citrus trees occurs in seasonal cycles or flushes 

throughout the year. The new shoots (NS) develop at the apical and lateral 

meristems of mature branches. Flushes are normally more intense during spring, 

less intense during early autumn, and absent in winter (Primo-Millo and Agustí, 

2020). Temperature and soil moisture are the main environmental variables that 

regulate flushing. Daily average ambient air temperatures lower than 12°C and 

higher than 35°C generally limit emergence of NS. Rainfall requirements are in 

the range of 600 to 1,300 mm yearly (Primo-Millo and Agustí, 2020; Davies and 

Albrigo, 1994). Solar radiation has an indirect role, since it interferes in CO2 

assimilation during photosynthesis (Davies and Albrigo, 1994). Major NS 

emergence in late spring and early summer, coinciding with increase in 

temperature and water availability, has been observed in São Paulo State (SPS) 

in Brazil (Carvalho et al. 2020; Oliveira, 2017; Garieri, 2016), Florida (Hall et al., 

2008; Hall and Albrigo, 2007) and California (Kistner et al., 2016) in the USA, in 

South Africa (Catling, 1969), and in Malaysia (Teck et al., 2011).  

Flushing also is related to the genetic interaction of the rootstock and 

scion. Rootstocks are widely used to control the size and vigor of citrus trees, 

thus affecting the vegetative growth of the scions growing on them (Bowman and 

Joubert, 2020; Spiegel-Roy and Goldschmidt, 1996). Oliveira (2017) evaluated 

flushing dynamics and NS intensity on six sweet orange (Citrus × aurantium var. 

sinensis L.) varieties grafted on ‘Swingle’ citrumelo [C. × aurantium var. paradisi 

ined. x Poncirus trifoliata (L.) Raf.], ‘Sunki’ mandarin (C. reticulata Blanco ‘Sunki’) 

and ‘Rangpur’ lime (C. × limonia Osbeck var. limonia), by counting the number of 

NS at variable time intervals, from November 2012 to December 2015, in central 

SPS. The work started when the trees were 1.5-year-old. The trees were growing 

in irrigated and non-irrigated areas. In the non-irrigated area, the number of NS 

were lower on ‘Hamlin’ sweet orange when grafted on ‘Sunki’ mandarin, and 

higher on ‘Valencia Late’ sweet orange grafted on ‘Rangpur’ lime. There were no 

differences between rootstocks within the irrigated area. Garieri (2016) evaluated 

flushing dynamics and intensity over time by counting NS on 10-year-old ‘Natal’ 

sweet orange grafted on ‘Swingle’ and six trifoliate (P. trifoliata) hybrid rootstocks, 

for 15 months in an irrigated area, also in central SPS. The assessments were 



25 
 

weekly in spring and summer and fortnightly in autumn and winter. Flushing 

patterns and NS intensities were similar for all rootstocks. 

Knowledge for the impact of the rootstock on vegetative growth of the 

canopy of citrus trees is important. It may be used to program planting density of 

new orchards, and subsequent cultural practices such as pruning and harvesting 

(Forner-Giner et al., 2014). It may also help to improve pest management 

programs, in particular, those involving the Asian citrus psyllid (ACP), Diaphorina 

citri Kuwayama (Hemiptera: Sternorrhyncha: Psylloidea), vector of Candidatus 

Liberibacter asiaticus (Las), the phloem-limited bacterium associated with the 

severe Asiatic form of huanglongbing (HLB), the most important disease of citrus 

(Bové, 2006). However, most efforts toward understanding flushing dynamics of 

citrus to improve ACP control and, consequently, HLB management, has involved 

rootstock hybrids and scion varieties with similar levels in growth and vigor. Also, 

the studies have not included the dynamics of individual NS growth and 

development on the canopy, and how NS is affected by the climate and the 

rootstock. The only dwarfing rootstock studied in relation to NS dynamics has 

been ‘Flying Dragon’ (P. trifoliata var. monstrosa (T. Itô) Swingle). When it was 

used as rootstock, a lower NS intensity was observed on ‘Valencia Late’, which 

coincided with lower HLB incidence as compared with the vigorous ‘Rangpur’ 

lime (Rodrigues, 2018). The incidence of citrus variegated chlorosis (CVC) also 

was lower on ‘Folha Murcha’ sweet orange trees growing on ‘Flying Dragon’, over 

a period of seven years after planting, as compared with other 11 vigorous 

rootstocks (Cantuarias-Avilés et al., 2011). As for HLB, CVC is also transmitted 

by sap-sucking insects feeding predominantly from xylem of immature flush 

growth (Dellapé et al., 2016). Lower incidences of the diseases were probably 

related to less than optimal incidence and growth of NS on the dwarfing rootstock 

and, thus, limited availability of suitable immature tissues for feeding and/or 

oviposition by the vectors.   

Given the influence of rootstocks, and scions, on the incidence and growth 

of NS and feeding and oviposition and transmission of Las by ACP, we carried 

out experiments involving nine rootstocks and four commercial scion varieties to 

determine potential impacts of rootstock-scion combinations on NS growth 

parameters favorable to ACP. Also, given the strong influence of the climate on 



26 
 

flushing dynamics and intensity, we explored possible correlations of flushing and 

NS growth with environmental factors for application in mathematical models.  

 

2. MATERIAL AND METHODS 

 

2.1 Experimental area and plant material 

 

The experiments were carried out between August 2017 and December 

2018 in Bebedouro municipality (20°53’16’’ S, 48°28’11’’ W; 600 m ASL), SPS. 

Climatic classification of this region is Cwa, characterized as tropical with dry 

winter based on the Köppen climate classification system (Rolim et al., 2007). 

Meteorological conditions were monitored daily using CR10 Measurement and 

Control System (Campbell Scientific Inc) located ca. 600 m away from the 

experimental blocks. The sequential water balance [water deficit (WD, mm) and 

actual evapotranspiration (AET, mm)] were calculated between assessment 

dates as proposed by Thornthwaite and Mather (1955) using an available water 

capacity of 100 mm. The potential evapotranspiration (PET, mm) was estimated 

using the FAO-56 Penman–Monteith (FAO PM) method (Allen et al., 1998). 

The experimental area consisted of four non-irrigated blocks of 0.8 ha, 

each corresponding to one scion variety: ‘Pera’ and ‘Folha Murcha’ sweet 

oranges, ‘Ponkan’ mandarin, and ‘Persian’ lime (C. × latifolia var. latifolia), which 

are broadly cultivated in Brazil (Fundecitrus, 2020). Each scion was grafted on 

nine rootstocks with contrasting growth patterns (Bowman and Joubert, 2020; 

Ramos et al., 2015) (Table 1). The trees were planted in February 2016, spaced 

2.0 m apart within rows and 5.0 m apart between rows resulting in approximately 

800 trees ha-1, and at 51° of azimuthal orientation for the rows. During the first 

evaluation date, canopy volume (V) of each tree was estimated using the 

formulae 𝑉 = (
2

3
) 𝜋𝑟2ℎ (Zekri, 2000), where r is the canopy radius and h is the 

tree height.  

 

 

 

 



27 
 

Table 1. Hybrids and commercial rootstocks categorized based on vigor (tree size), 
which were used with ‘Pera’ and ‘Folha Murcha’ sweet oranges, ‘Ponkan’ 
mandarin and ‘Persian’ lime scions. 

Rootstock common name Scientific name/ parentage description Vigor classification 

‘Rough’ lemon C. × limonia var. jambhiri ined. vigorous 

‘Sunki’ mandarin C. reticulata ‘Sunki’  vigorous 

‘Swingle’ citrumelo C. ×aurantium var. paradisi x P. trifoliata intermediate 

‘BRS Bravo’ 
C. reticulata ‘Sunki’ x (C. × limonia var. limonia x P. 

trifoliata) - 059 
intermediate 

‘BRS Cunha Sobrinho’ 
C. reticulata ‘Sunki’ x (C. × aurantium var. paradisi 

× P. trifoliata) - 041 
intermediate 

San Francisco C. reticulata ‘Reshni’ x P. trifoliata ‘Swingle’ - 287 intermediate 

Los Angeles C. reticulata ‘Reshni’ x P. trifoliata ‘Barnes’ - 245 dwarfing 

‘BRS Matta’ 
C. reticulata ‘Sunki’ x P. trifoliata var. montrosa 

‘Flying Dragon’ - 006 
dwarfing 

‘Flying Dragon’ trifoliate Poncirus trifoliata var. monstrosa dwarfing 

 

During the entire evaluation period, the trees remained unpruned and 

received the standard management practices recommended for citrus crops in 

SPS (Carvalho et al., 2005). To reduce ACP populations, during the first three 

years of planting, systemic insecticides were applied via drench, three to four 

times per year plus contact insecticides sprayed on tree canopies, at every 10-

day interval, which was the only insect control measure after the third year.  

 

2.2 Flushing dynamics and intensity 

 

Every 20 days, the new shoots (NS) found within the projection of a square 

frame of 50 cm x 50 cm, positioned in the outer surface of the canopy, at ca. 1.5 

m above ground, were counted and classified by their respective developmental 

stage (Cifuentes-Arenas et al., 2018). Eight apparently healthy trees per 

scion/rootstock combination, randomly distributed within the orchard, were 

selected. Each tree served as a replication. This number of trees was previously 

determined as the minimum number that would result in a sampling error of 10%, 

(Carvalho et al., 2020). The 20-day assessment interval was chosen because it 

is shorter than the time required for a given NS to develop from bud swelling and 

initial growth (stage v1) to complete maturation (stage v6), which takes 35 to 45 

days at 24 to 26oC. Stage v1: from bud swelling to initial bud emergence. Stage 



28 
 

v2: initial stem elongation with all margins of the leaves closed. Stage v3: initial 

leaf blade expansion with margin of lower leaves opening. Stage v4: unfolding of 

all leaves and final leaf number defined. Stage v5: leaves fully expanded, green-

light yellow coloured and gradual hardening from top to the base. Stage v6: 

vegetative shoot completely matured with leaves fully hardened and green-dark 

(Cifuentes-Arenas et al., 2018). Along with NS counting, the intensity of NS for 

the whole period of assessment for each sampled tree was estimated also based 

on the area under flush shoot dynamics (AUFSD), using the formula: 

𝐴𝑈𝐹𝑆𝐷 = ∑ (
𝑁𝑆𝑖+1 + 𝑁𝑆𝑖

2
) ∗ (𝑇𝑖+1 − 𝑇𝑖)

𝑛−1

𝑖=1

 

where NSi is the number of NS in stages v1 to v6 in the ith day observation, and 

Ti+1-Ti the number of days among observations (Campbell and Madden, 1990).  

 

2.3 New shoot growth dynamics  

 

The dynamics of NS growth was assessed during the spring of 2018 

(October to November) and the autumn of 2019 (April to May). Three of the nine 

rootstocks used in the study of flushing dynamics were selected: ‘Rough’ lemon 

(vigorous), ‘Flying Dragon’ trifoliate (dwarfing) and ‘Swingle’ citrumelo 

(intermediate). Four trees per each scion/rootstock combination were used. To 

stimulate flushing, three to six branches of each tree were pruned to remove 5 to 

10 cm of the apex of the branches, on both sides of the central portion of the 

canopy. Three to five leaves just below the pruning sites also were removed. 

Approximately 12 to 13 days after pruning, three apical NS at the early v1 growth 

stage were tagged on each side of the canopy. The length of the NS was 

measured every three to four days using a digital caliper (Zaas Precision, 

Amatools Commercial Ltda, Piracicaba, SP, Brazil). The evaluations proceeded 

until the NS reached the v6 stage. For this study, non-linear regression models 

were applied to the accumulated degree-days (DD) needed for the NS to reach 

the assessed length at every evaluation date, from the pruning day up to v6 stage. 

DD is a measure of heat accumulation and was estimated as follows: 

𝐷𝐷 = ∑ 𝑇𝑚𝑒𝑑𝑖

𝑛

𝑖=1

− 𝑇𝑏𝑎𝑠𝑒 



29 
 

where ΣDD is the sum of growing degree-days, Tmed the absolute daily mean air 

temperature (°C) at the ith day observation, and Tbase the lower temperature limit 

for the shoot to grow (Allen, 1976), which was determined for citrus trees as 9°C 

by Cifuentes-Arenas (2017). 

 

2.4 Data analysis 

 

2.4.1 Flushing dynamics and intensity  

 

Before analyses, all data were assessed for homoscedasticity (Levene, 

1960), and normality (Shapiro and Wilk, 1965). Box-Cox transformation of the 

data was performed when necessary (Osborne, 2010). A split-plot analysis of 

variance for repeated measures overtime was applied to the number of NS for 

each scion separately, in a fully randomized design. The rootstocks comprised 

the main plot, the assessment date the subplot, and the eight trees the 

replications. For analysis, the 24 assessment dates were grouped in every three 

consecutive dates, which corresponded to eight periods of distinct seasonal 

climatic conditions. AUFSD for the whole assessment period was calculated for 

each sampled tree. The evaluations proceeded on the same trees from the first 

to the last evaluation date. When significant differences were detected, the Scott-

Knott test was applied (P < 0.05).  

In order to assess the influence of the environment variables on flushing 

dynamics and intensity, multiple linear regression was performed with the number 

of NS as the dependent variable and the daily average values of maximum (Tmax), 

minimum (Tmin) and average (Tavg) temperature (°C) and accumulated rainfall 

(mm), taken during the 20, 40 and 60 days prior to the evaluation date, as the 

independent variables. The best fit final model was selected based on Akaike 

Information Criterion (AIC). The ‘stepwise backward’ procedure (P < 0.05) was 

used to select the significant independent variables.  

 

2.4.2 New shoot growth 

 

The data on NS growth as a function of DD was subjected to non-linear 

regression analysis. The models tested included the sigmoid, 𝑌 =



30 
 

𝑌𝑎𝑠𝑦𝑚𝑒𝑥𝑝{−𝑒𝑥𝑝[−𝑘(𝑡 − 𝑡𝑚)]} (Gomperz, 1825), and the logistic, 𝑌 = 𝑌𝑎𝑠𝑦𝑚/{1 +

 𝑒𝑥𝑝[−𝑘(𝑡 − 𝑡𝑚)]} (Verhulst, 1838) functions, where Y is the estimated shoot 

length, Yasym the estimated asymptotic Y value, t the time from bud swelling to 

shoot maturity, calculated in DD, tm the inflection point at which growth is 

maximized, and k the NS growth rate. The final model was selected based on the 

AIC (Akaike, 1998). Pearson correlation analyses were performed to assess 

relationships among tree height, tree diameter and canopy volume using AUFSD 

and shoot final size. 

Split-plot analysis of variance and Pearson correlation analysis were 

performed using the statistical software R, version 3.6.1 (R Development Core 

Team 2015). Regression analyses were performed using Python language 

(Anaconda Python, Continuum Analytics) (Van Rossum and Drake Jr, 1995).  

 

3. RESULTS 

 

3.1 Flushing dynamics and intensity 

 

When the evaluations started, the trees of different scion and rootstock 

combinations varied in height and canopy volume as indicated (Figure 1).  

 

 

Figure 1. Average tree height (m) and canopy volume (m3) of Pera’ and ‘Folha 
Murcha’ sweet oranges, ‘Persian’ lime and ‘Ponkan’ mandarin grafted on 

3.12

1.80

3.86

1.68

0.40 0.38
0.76

0.24

0

1

2

3

4

Pera Folha Murcha Persian Ponkan

C
a

n
o

p
y
 v

o
lu

m
e

 (
m

3
)

Vigorous

Dwarfing

1.80

1.53

1.88
1.70

1.30 1.25
1.40 1.33

0

1

2

Pera Folha Murcha Persian Ponkan

T
re

e
 h

e
ig

h
t 
(m

)



31 
 

vigorous ‘Rough’ lemon and dwarfing trifoliate ‘Flying Dragon’ rootstocks 
measured August 2017 in Bebedouro, SPS. Standard errors – indicate 
Tree height (m) ± se, Canopy volume (m3) ± se on X axis.  

 

Over the evaluation period, the highest and lowest values of solar 

radiation, evapotranspiration, maximum and minimum absolute daily 

temperatures were, respectively, 28.0 and 15.5 MJ m–2 day–1, 4.6- and 0.7-mm 

day–1, 37.3°C and 15.7°C and 22.2°C and 6.2°C. The accumulated rainfall was 

1,690mm. More detailed information on air temperature and rain is shown in 

Figure 2.  

Although the interaction of rootstocks and assessment periods was 

significant for all scions, which indicates that the same environmental condition 

interacted differently depending on the scion and rootstock combination 

(Supplementary Table S1), the overall flushing patterns were similar for all 

combinations (Figure 2). 



32 
 

 

Figure 2. Patterns of new shoot occurrence and intensities on the canopy of 
A) ‘Pera’ sweet orange, B) ‘Folha Murcha’ sweet orange, C) ‘Persian’ lime and 
D) ‘Ponkan’ mandarin grafted on all nine rootstock hybrids (see Table 1); E) 
Water balance [water deficit (red area) and excess (blue area), in millimeters] 
during the assessment period; F) Accumulated rainfall (blue bars) and 
maximum (■), average (▲) and minimum (♦) air temperature registered in the 
periods between sampling dates (each 20 days), from August 2017 to 
December 2018 in Bebedouro, SPS. The arrows indicate the first and last 
assessment dates.   
 

 

0

10

20

30

0

10

20

30

0

10

20

30

0

50

100

150

200

250

300

350

400

0

5

10

15

20

25

30

35

40

R
a

in
fa

ll 
(m

m
)

A
ir

 T
e

m
p

e
ra

tu
re

 (
°C

)

Date

A

B

C

0

10

20

30 D

S
o

il
w

a
te

r
a

v
a

il
a

b
e

l
(m

m
)

A
ir

 t
e

m
p

e
ra

tu
re

(°
C

)

R
a

in
fa

ll
(m

m
)

E

F

N
e

w
 s

h
o

o
ts

p
e

r 
m

2
A

ir
 t

e
m

p
e

ra
tu

re
( 

C
)

A
c

c
u

m
u

la
te

d
ra

in
fa

ll
(m

m
)

‘Persian’ lime

‘Pera’

‘Folha Murcha’

‘Ponkan’



33 
 

Rootstocks affected flushing dynamics on ‘Pera’, but only during the 2nd, 

4th and 7th assessment periods was the number of NS statistically different (Table 

S2). ‘Pera’ on ‘Rough’ lemon produced major flushes in the 2nd and 4th assessment 

periods, but no statistical differences were found compared with those on other 

vigorous or intermediate rootstocks. On ‘BRS Bravo’, ‘Pera’ produced higher 

number of NS in the 7th assessment period, but it was not statistically different 

from that on ‘Sunki’ (Figure 3).  

Flushing intensity was also evaluated through the area under flush shoot 

dynamics (AUFSD), for each scion variety separately. This method simplified the 

analysis, since it combines the intensity and frequency of the NS in a single value. 

‘Rough’ lemon on ‘Pera’ induced the highest flushing intensity, but it did not differ 

from ‘Sunki’, ‘Swingle’, ‘BRS Bravo’ and ‘BRS Cunha Sobrinho’. AUFSD was 

however 1.9 times greater on the vigorous ‘Rough’ lemon than on the dwarfing 

‘Flying Dragon’ rootstock (Figure 3).  

 

1139 b

2168 a

2096 a

1730 a

2005 a

1794 a

1613 b

1454 b

1434 b

0

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30

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30

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30

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30

Aa Ba Cb

Aa

Ba Cc

Bc

Bb Cb

ACD D D

B A B B A

C B C C A

C B C C A

A A B B A

B A B B A

C B C C A

C B C C A

C B C C A

Aa Aa

Aa

Aa Ba Aa

Aa Aa Ab

Aa Aa

Bb

Bb

Cc

Cc

Cc

Bb

Cb

A

B

C

D

E

F

G

H

I

1st 2nd 3rd 4th 5th 6th 7th 8th

N
e

w
 s

h
o

o
ts

p
e

r 
m

²

AUFSD



34 
 

Figure 3. Patterns of new shoot occurrence and intensities and area under 
flush shoot dynamics (AUFSD) on the canopy of ‘Pera’ sweet orange 
trees grafted on: A) ‘Rough’ lemon; B) ‘Sunki’ mandarin; C) ‘Swingle’ 
citrumelo; D) ‘BRS Bravo’; E) ‘BRS Cunha Sobrinho’; F) San 
Francisco; G) ‘BRS Matta’; H) Los Angeles; I) 'Flying Dragon' 
trifoliate. Lowercase letters compare means among rootstocks within 
each assessment period, and uppercase letters compare means 
among assessment periods within each rootstock. Means followed by 
the same letter belong to the same group by the Scott-Knott’s Test (P 
< 0.05).  

 

Rootstocks affected flushing dynamics on ‘Folha Murcha’ in the 1st, 2nd, 

4th, 7th and 8th assessment periods (Table S2). On ‘Sunki’ and ‘BRS Bravo’, ‘Folha 

Murcha’ produced higher number of NS in the 1st assessment period. On ‘Flying 

Dragon’, ‘Folha Murcha’ produced lower number of NS in the 2nd assessment 

period. In the 4th assessment period, major flushes were detected on trees 

growing on ‘BRS Bravo’, ‘Sunki’, San Francisco and ‘Rough’ lemon. On ‘Rough’ 

lemon, ‘Sunki’, ‘BRS Matta’ and ‘Swingle’, ‘Folha Murcha’ produced higher 

number of NS in the 7th assessment period. On ‘Rough’ lemon, ‘Folha Murcha’ 

produced a higher number of NS in the 8th assessment period, but it was not 

statistically different from that on ‘Swingle’ (Figure 4). 

Highest AUFSD was found on ‘Folha Murcha’ grafted on ‘Rough’ lemon. It 

did not differ from that on ‘Sunki’ but was 3.1 times greater than that on ‘Flying 

Dragon’ (Figure 4). There was no variation in the number of NS overtime on 

‘Folha Murcha’ on ‘Flying Dragon’ (Figure 4) (Table S3). 

 



35 
 

 

Figure 4. Patterns of new shoot occurrence and intensities and area under 
flush shoot dynamics (AUFSD) on the canopy of ‘Folha Murcha’ 
sweet orange trees grafted on: A) ‘Rough’ lemon; B) ‘Sunki’ 
mandarin; C) ‘Swingle’ citrumelo; D) ‘BRS Bravo’; E) ‘BRS Cunha 
Sobrinho’; F) San Francisco; G) ‘BRS Matta’; H) Los Angeles; I) 
'Flying Dragon' trifoliate. Lowercase letters compare means among 
rootstocks within each assessment period, and uppercase letters 
compare means among assessment periods within each rootstock. 
Means followed by the same letter belong to the same group by the 
Scott-Knott’s Test (P < 0.05). 

Rootstocks influenced flushing dynamics on ‘Persian’ lime, but only during 

the 1st, 2nd and 4th assessment periods the number of NS were different (Table 

S2). On ‘Swingle’, ‘Persian’ lime produced higher number of NS in the 1st 

assessment period but was not statistically different from that on San Francisco. 

On ‘Rough’ lemon, ‘Persian’ lime produced higher number of NS in the 2nd 

assessment period but were not statistically different from those on San 

Francisco and ‘BRS Cunha Sobrinho’. ‘Rough’ lemon induced higher NS in the 

2553 a

AUFSD

2311 a

2034 b

1950 b

1443 c

1546 c

1655 c

1375 c

0

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AaBaDb Ba Ca

Aa Aa Ab

Cb Ba Cb Ba Aa

Aa Aa Aa Ab Ab

Bb Aa Bb Bb Bc

Ab Aa Aa Bb Ab

Bb Aa Bb Aa Ab

Bb Aa Bb Bb Ab

b b b b c

B D D

Aa Aa A B B

B C C

A B B

B C C

A B B

A B B

B B B

1st 2nd 3rd 4th 5th 6th 7th 8th

A

B

C

D

E

F

G

H

I

813 d

N
e
w

 s
h

o
o

ts
p

e
r 

m
²



36 
 

4th assessment period. ‘Rough’ lemon, ‘Sunki’ and San Francisco induced major 

overall flush intensity (Figure 5).   

 

 

Figure 5. Patterns of new shoot occurrence and intensities and area under 
flush shoot dynamics (AUFSD) on the canopy of ‘Persian’ lime trees 
grafted on: A) ‘Rough’ lemon; B) ‘Sunki’ mandarin; C) ‘Swingle’ 
citrumelo; D) ‘BRS Bravo’; E) ‘BRS Cunha Sobrinho’; F) San 
Francisco; G) ‘BRS Matta’; H) Los Angeles; I) 'Flying Dragon' 
trifoliate. Lowercase letters compare means among rootstocks within 
each assessment period, and uppercase letters compare means 
among assessment periods within each rootstock. Means followed by 
the same letter belong to the same group by the Scott-Knott’s Test (P 
< 0.05). 

 

Rootstocks affected flushing dynamics on ‘Ponkan’ in the 2nd, 3rd and 4th 

assessment periods (Table S2). On ‘Rough’ lemon, ‘Ponkan’ produced higher 

number of NS in the 2nd and 3rd assessment periods. On ‘Flying Dragon', ‘Ponkan’ 

produced significantly lower NS in the 4th assessment period. Overall, ‘Rough’ 

2134 a

1803 a

1554 b

1536 b

1486 b

1768 a

1349 b

1416 b

1249 b

0

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Aa AaCb

Bb Ab Ab

Aa Ab Ac

Bb Ab Ac

Bb Aa Ac

Ba Aa Ab

Bb

Cb

Cb

Ab

Bb

Ab

Ac

Bc

Bc

B C C B A

A C C A A

A B B A A

B B B A A

B C C B A

C D D C A

B C C B A

B C C B A

B C C B A

1st 2nd 3rd 4th 5th 6th 7th 8th

A

B

C

D

E

F

G

H

I

AUFSD

N
e
w

 s
h

o
o

ts
p

e
r 

m
²



37 
 

lemon induced 3.2 times more NS than ‘Flying Dragon’ (Figure 6). There was no 

variation in the number of NS overtime on ‘Ponkan’ on ‘Flying Dragon’ (Figure 6) 

(Table S3). 

 

 

Figure 6. Patterns of new shoot occurrence and intensities and area under 
flush shoot